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SOME  METHODS  OF  STUDYING  THE  METABOLISM 
OF  HIBERNATING  ANIMALS 

BY 

MYRON  THOMAS  TOWNSEND 
B.  S.,  Bates  College 
1918 


THESIS 

Submitted  in  Partial  Fulfillment  of  the  Requirements  for  the 

Degree  of 

MASTER  OF  SCIENCE 
IN  ZOOLOGY 

IN 

THE  GRADUATE  SCHOOL 

OF  THE 

UNIVERSITY  OF  ILLINOIS 
1921 


Digitized  by  the  Internet  Archive 
in  2016 


https://archive.org/details/somemethodsofstuOOtown 


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TABLE  OF  CONTENTS. 

I  Introduction:  the  nature  of  the  problem; some  advan- 


tages of  toads  as  experimental  animals 1 

II  Materials. Methods  of  collecting  and  keeping  toads 2 

A.  Scarcity  of  animals  during  the  autumn 3 

B.  Florida  toads 3 

C.  Collecting  during  the  spring  near  a breeding  pond.  . 4 

D.  The  outdoor  cage  without  water  supply 5 

E.  The  flower-pot  type  of  cage 6 

F.  Use  of  the  large  aquarium 6 

0.  The  toad  pen  in  the  greenhouse ; methods  of  feeding.  . 6 

III  The  water  content  of  the  toad 8 

IV  The  absorptive  force  exerted  by  the  toad's  skin JO 

A.  Previous  work  on  the  frog's  skin 10 

B.  Description  of  the  osmometers  used 11 

C.  Methods  of  recording  results 13 

D#  Experiments  with  toad's  skin 13 

E*  Conclusions 17 

V Respiration  apparatus 20 

A.  Apparatus  for  obtaining  pure  air 20 

B.  Apparatus  for  determining  CO2  by  titration 22 

C.  The  respiratory  chamber. 23 


D.  Experiments  with  the  titration  apparatus 25 

E.  A modified  biometer  for  animals  as. large. as  toads  . 26 


F.  Small  respiratory  chamber  for  insects, etc 31 

VI  Summary 33 

VII  G-raphs, plates  and  tables 35 

VIII  Bibliography A9 


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I Introduction 

Hibernation  has  been  studied  by  various  workers, and  much 
attention  has  been  given  to  hibernating  mammals  in  the  hope  of 
finding  some  internal  physiological  explanation  for  the  phe- 
nomenon, More  recently  emphasis  has  been  laid  on  the  condition 
as  observed  in  insect  pests  and  its  bearing  upon  methods  for 
their  control.  A great  many  phases  of  the  subject  have  not  yet 
been  studied, and  especially  is  this  true  of  certain  ecological 
relations  to  external  environment  as  opposed  to  the  internal 
physiological  side  of  the  problem.  It  is  my  purpose  to  study 
the  phenomenon  of  hibernation  from  an  ecological  point  of  view 
. with  the  hope  of  throwing  new  light  on  some  of  the  economic 
or  purely  scientific  problems  involved, especially  as  regards 
this  condition  in  cold  blooded  animals. 

With  this  in  vie?/  it  seemed  best  at  the  outset  to  choose 
an  animal  for  experimentation  large  enough  so  that  various 
d-eterminations  such  as  body  temperature, blood  analyses , etc . 
could  be  made  if  desirable. The  common  toad,Bufo  americanus  Le 
Conte, seemed  to  fit  these  requirements  very  well, being  a land 
animal  during  the  greater  part  of  its  Ilf e-history, cold  blooded, 
accustomed  to  pass  the  winter  in  a state  of  hibernation,and  large 


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enougji  to  admit  of  easy  determinations  of  body  temperatures, 
blood  analyses,  etc.  Accordingly,  the  first  part  of  the  paper 
is  given  over  to  a study  of  various  methods  of  keeping  toads  in 
captivity  for  experimental  purposes.  In  the  course  of  this 
study,  the  importance  of  the  water  content  of  the  animals  became 
evident  (Part  3)  and  led  to  some  preliminary  investigation  as  to 
the  regulatory  mechanism  involved,  the  results  of  v/hich  are  dis- 
cussed in  Part  4.  The  remainder  of  the  paper  is  occupied  with 
descriptions  of  apparatus  for  studying  the  carbon  dioxide  output 
of  the  animals  together  with  some  preliminary  experiments  v/ith 
these.  The  data  so  far  gathered  is  not  sufficient  to  warrant 
the  drawing  of  any  new  conclusions  regarding  the  metabolism  of 
hibernating  animals,  but  such  data  will  be  gathered  later  when 
the  apparatus  and  methods  here  described  have  been  used  to  a 
greater  extent. 

II.  Materials 

In  the  fall  of  1919,  I began  my  work  on  toads  by 
attempting  to  collect  specimens  in  the  vicinity  of  Urbana,,  111. 
At  that  season  of  the  year  I foimd  tliem  to  be  very  scarce,  and 
after  two  or  three  v/eeks  of  persistent  searching  I had  procured 
only  tv/o  or  three  toads  and  a few  snakes.  Later  observations 
indicated  that  the  toads  must  have  been  in  the  vicinity,  but  a.t 
that  season  they  were  widely  scattered  and  well  hidden  so  that 
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numbers.  Accordingly  a dozen  Florida  toads  were  procured  from 
tlie  Chicago  Biological  Supply  House.  They  were  shipped  in  a 
screen-sided  wooden  box  half  full  of  oak  leaves,  presimably  from 
their  native  habitat. 

The  toads  and  leaves  were  transferred  to  a wire  screen 
cage  ( 14  in.  by  10  in.  by  8 in.)  and  placed  in  the  Vivariimi 
green-house.  The  cage  was  placed  on  strips  of  v/ood  above  a water 
table  and  a continuous  stream  of  water  was  kept  flowing  under  it 
to  keep  the  air  moist.  The  temperature  of  the  room  was  about  68° 
F.  About  the  middle  of  December,  upon  examining  the  cage,  I 
found  one  toad  dead,  and  I then  observed  that  while  the  water  ran 
under  the  cage  continually,  the  oak  leaves  immediately  surround- 
ing the  toads  were  quite  dry  and  brittle.  I lowered  one  end  of 
the  cage  into  the  water  and  about  an  hour  later  found  all  the 
toads  congregated  in  that  end  and  several  of  them  swelled  up  to 
a considerable  size  because  of  the  amount  of  water  which  they 
had  imbibed.  This  experience  served  to  emphasize  the  importance 
of  a readily  available  water  supply  for  keeping  toads  in  a 
healthful  condition,  and  to  meet  this  need  better,  I removed 
the  toads  to  a new  location.  I returned  the  animals  to  the 
screen-sided  box  and  placed  this,  screen  side  down,  on  top  of 
a galvanized  iron  tank  of  water.  The  rest  of  the  tank  was 
covered  by  a board.  By  this  means  I hoped  to  obtain  a suffi- 
cient degree  of  saturation  of  the  air  in  the  box  to  keep  the 
toads  in  a healthful  condition.  The  whole  enclosure  was  placed 
on  a brine  coil  in  a cold  room  (temperature  about  11°  C.  ) in 


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order  to  lower  the  rate  of  metaho lism  of  tlie  animale  and  keep 
them  hihernating  if  possible.  In  this  inclosure  the  toads  re- 
mained apparently  dormant,  but  on  Jan.  5,  1920,  I found  that  two 
more  had  died  and  from  this  time  on  tliey  slowly  died  off  until 
at  about  the  middle  of  the  month  all  were  dead.  The  leaves  in 
the  cage  at  this  time  were  observed  to  be  quite  dry  which  would 
seem  to  indicate  that  the  air  in  the  box  was  not  saturated  with 
moisture  as  much  as  I had  hoped  it  would  be. 

Some  field  observations  during  the  spring  of  1920  fur- 
ther emphasized  the  importance  of  moisture  in  the  environment 
of  the  toad.  At  this  time  I began  to  collect  animals  from  the 
vicinity  of  the  lily  pond  on  the  south  campus.  Here  the  toads 
gather  each  spring  to  breed  and  lay  their  eggs  in  the  pond.  One 
or  tv/o  cold  nights  occurred  late  in  the  spring  and  I found  that 
at  such  times  the  toads  sought  shelter  in  various  places,  and 
quite  a number  of  them  fell  into  the  concrete  pits  in  front  of 
the  cellar  windows  of  the  green-houses  near  by.  These  pits  are 
about  three  feet  deep  and  four  feet  wide  and  covered  by  open 
iron  gratings  thru  which  the  toads  easily  fell.  The  windows  into 
the  cellar  were  kept  closed  and  the  toads  were  thus  easily 
trapped.  The  bottoms  of  these  pits  were  usually  found  covered 
with  a layer  of  leaves  and  soil  in  which  the  animals  burrowed, 
and  it  was  observed  that  v/hen  the  leaves  and  soil  dried  up,  the 
toads  buried  in  them  v/ere  also  found  to  be  dried  up  and  dead. 

If  the  leaves  and  soil  in  a window  pit  were  found  damp  usually 
from  one  to  ei^t  or  ten  healthy  toads  could  be  found  by  over- 


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\iX>Vnimu  bs^Qli  pY'?'*#  rfXq  Vdolfliv  J1  kt  \t . 


'JJiklfhiaM 


f ''»%  ^ *'  ' J M ^ K* 

•|W  '.'I  - _ '.1.  -AV  W V.r*A(  <4l«  ^ 1 

' > ■.'  -it 


i' 


Ik  « A # i 


-5- 


turning  the  leaf  mulch.  In  this  way  about  50  specimens  were 
collected  and  brou^t  to  the  vivarium,  where  several  methods  of 
keeping  them  alive  were  tried,  as  follows  - 

Three  toads  v/ere  placed  in  a screen  cage  ( 12  in.  by 
12  in.  by  12  in.  ) and  this  was  placed  on  the  surface  of  the 
ground  in  a sheltered  corner  on  the  north  side  of  the  vivarium. 
No  sunlight  ever  reaches  the  spot  and  the  soil  is  always  moist. 

A shallow*  depression  was  dug  in  the  soil  and  the  cage  was  placed 
in  this  and  banked  up  about  two  inches  on  all  sides  with  moist 
earth.  A few  leaves  were  placed  in  the  cage.  Although  the 
bottom  screen  of  the  inclosure  was  all  that  intervened  between 
the  toads  and  the  moist  surface  of  the  earth,  and  the  air  in  the 
inclosure  mi^t  be  expected  to  be  reasonably  moist  and  cool, 
nevertheless  a fev/  days  later  the  animals  were  found  to  be 
stretched  out  on  the  cage  floor,  too  stiff  to  allow  ready  move- 
ment of  the  limbs.  They  were  removed  to  the  laboratory  and  held 
under  a cold  water  faucet  and  two  of  them  responded  by  a very 
slight  movement.  Repeated  drenching  v/ith  water  completely  re- 
vived these  two  and  they  were  apparently  as  healthy  as  before 
their  confinement.  The  third  toad  did  not  recover.  Apparently 
the  moisture  content  of  his  body  had  been  reduced  below  the  min- 
imum. The  few  leaves  contained  in  the  cage  were  found  to  be  par^ 
tially  dried  up  when  the  animals  were  removed. 

The  foregoing  observations  would  seem  to  indicate  that 
the  toad  must  be  able  to  come  into  direct  contact  with  moist 
soil,  perhaps  to  burrow  in  it  in  order  to  maintain  the  proper 


n • 


,t  , ■ • . . ,•■• . « , X 

11  cnovi  :f*  oi  \.'w-  .#4,! -.‘A  >x  " .Hftfxa.ti^fx  »4i 

/.',  .. ..  ^ V- > ,.  ■ ' w „■*  ■ 


• W ‘ LJ  ■,•  - ■■  , ■ • . , , . 

J iihru'Tj.^.  Su'x  rVn  ..JWlTtiiiiiv  .fi  i 4*  f>#/  >ii 


j 


‘■5  '■  , ■ ■'  " 

c^tf¥ 


1 «|icv  r;if.N'-.'4?  , oi  ^ilft 
, ftrf.*  lo  n,ki^  1x9  lor,<kt;t' ; >(11  / C''*’ . P\1 

. * A ^ ^ 

^ Iti  »■!  r.  .•»,  i-.M.oxi-'  iio  It.,.':', 4*  lU 

;j'l|ir^  C*4^rl..  . |/  rr.*  S>^i.  5u>  ^ ^'/t5  ^i^lj-..' 4 j 

■ '*  ’ ' ' '"-X*  " [ ' 

^^:v  aj.44 r^-;  -^r:7  tfs*  J 

L./'  „ '■  •fl.®  ■ *^  ■''  I "" 

J »,  o.  tPil  hAiff,  ' 

U ,*  ' '.  I '* 

rija:  n«  n*^vU.f  ®T‘^ 


!l  tir;  //C  >r  ii4 


a?.4pi^'  fjJW 


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o.*  D%j^  .rt'TKi**:  & v4  > 

|A  0»,V4:>-  o«i  ,'rto/v»  e-u-.r^ ii 

'vfc  xf0^4lvX4Ui'  '^>-  ' M .‘.ri>L-Vjt  r.  Sih^  . 

» 4 * : . I . . V . - '.A  *)  ! « 


l^ 


“ J 


Ifirxr  4:.  iiijr  ioa'-ti  d /O  i^  cmi  x>o^«s1t 

■ .•'  '.I  I '/  I « 1 


►■■  ■•'.  "'S''.  * • 'M  ' 

|f.iv  -9^  vl^' I'j  'i*;?  tf/iw  ^ ’ 

I . <1  •►  v'  . _ '^  .'"  r ” ‘ 

» ■ . ,»^  .V  V.:,  .-  a-.'^',  ''•  -4 


4fe  ^ M 

■•  i> . 


• . " . ; .V  V..: . . j ■ . r . ‘ ' . .>  fi\m 

^ '-v/n  P/t5  WcCfftf  -»44^  W"' 

^*' .,  ^ , * ''•'*  i-  - -'•*  yx  /■  *T4'i  -r  ‘ ',  *i  ••  Si 


•«-,st  ^ 05>  bvi\cA  o’xtv..,  ofiij  rJi  .;» .oJL-  /■:':•  « 

' 'h!  ’-4  ^v**,  ' I ,■«  . ' ' ,’,  , *i,  r 

r •■  '...  J.'  . . .,  y.  J-  .,  . M^J 


;^$viL-frit,i;  n [j^itw  ' cuu'  aul  ^i*i4  _. 

*1  ■ j 7 * 1.  ^ ' ,-  jT  t ■ ^ t 

00 tifjf'fft  5^f  0?  it 

I ifl  4i  », Clift  1^ 

y A -'  ' ./  j,  ; _ . ■ ,ut..’.  .*. . hIi 

aww  ».  ij-,. ^ •>  ■ ,..•  ■ . ,s  . r ' ' ■:  mil 


-6- 


moisture  content  necessary  for  health.  This  conclusion  is  fur- 
ther borne  out  by  experiments  with  another  type  of  cage  as 
follows: 

Several  flowerpots  were  prepared  for  use  as  inclosurss; 
{See  Plate  I,  Pig.  l) ; each  was  filled  2/3  full  of  soil,  pro- 
vided with  a wire  screen  cover  and  set  in  a saucer  of  water.  The 
soil  in  the  pot  soon  absorbed  a large  amount  of  water  and  became 
of  the  consistency  of  v/et  mud.  One  or  two  toads  v;ere  placed  in 
each  pot  and  the  covers  weighted  down  to  prevent  their  escape. 

In  this  type  of  cage  so  far  as  I have  tried  it,  the  toads  live 

in  perfect  health.  Usually  the  animal  burro7/s  backward  into  the 
mud  and  sometimes  completely  buries  itself  out  of  sight  by  this 
means.  When  dug  up  the  toads  were  always  fat  and  plump  and  v/ide 
awake.  Apparently  their  surroundings  of  wet  mud  furnished  just 
the  right  kind  of  environment  for  them. 

A number  of  toads  were  kept  in  a large  aquarium  tank 

with  about  two  inches  of  water  in  the  bottom  and  provided  with 

scattered  bricks  and  boards  for  them  to  climb  onto.  Here  they 
remained  apparently  in  good  health.  Several  occupied  a pile  of 
v/et  soil  in  one  end  of  the  tank,  burrowing  deep  in  the  mud,  fre- 
quently in  pairs,  the  male  clasping  the  female. 

As  a means  of  keeping  the  toads  thru  the  summer,  I 
arranged  an  inclosure  in  an  open  screen-covered  room  connected 
with  the  green-house.  A corner  of  the  room  underneath  some 
tables  v/as  fenced  off  with  vrire  screen  two  feet  hi^.  A pile  of 
black  dirt  was  dumped  on  the  concrete  floor  in  the  middle  of  the 


ttixCT  .-^".Litmii  t«it  xt«0V 

|-  li'J.  ‘ ■ , .;.  K 


V 


iv^'liP  t’.'/  f'cri.'tj^a  v*.<^  Ji/fli  •rfl.'ij  jI 

* t '*^W  ' 


v^-  • i;  'it . .-jc  ■:  '-iC-':  ■- 


Mif  'L  ? 0O>MV.  ' 1 4r“rcVbe  ‘’* 


- '••T  *'  . -X  . --I  J iH'y  C\;.  «-i  * ¥'.9'*  ‘ rfj)-  f»'.  '’(,  i'’i  J--  a“’ 


.:<iX.%v  \C  4.  /.Jt,  -oft  eiiv*  « 


i,’  , tMjT 


r-  Javog^  i)f'Ji-*i  A ' ■ 4 i<i.,T  a.:x  cl‘ 

^ . ^^*aS  .Tip 

0**-  *0  i*r.C'  J*>V  *♦»;' 

,i;  ^^lTC  • . I *.  -i  1 o»  'rc)  nc^3  A^fU  oT 

^'  . ' ’ ’ ''t,  ' ■ ' ■ > 

t.  * .V  ' t . , . ^JXJ|!^4  ^Q!‘V;%a  ni  i 

f .-r  ’■■'•■  ,1’'  i'Vl 

, i)i-i  i wit^  ^w«  n-i-''-'  aoifl  ■ 

t ■"  . " ' ■ ■•  ma 

x-Lt  u.4?t  :,i /iC-r?Wvr  --  : * v : ,.;*■<>  ••n^^ 


I 


• t'J-  '‘*ol  p*flv  ,•*#:»  iwVa.',l^ 


W(# 


^ ityii-.iii/*  .1  ^.nX  i<^3  lE^  i#W3tx;,t  • 

‘ **  ' ' 4 ■ 


p^,  , -»i^r  wf 41'i'  cm^  'Jrao^i  ^i  v iif 

k'..  ' . - / . _.  •■ . • ' / k'  'f  • ;1 


‘^  ’-  ;v.t  'jrsal'  - .y^io  .’ar^Xo  u#  coirtil  seV  biui,  BS?oA<<.6&'isJ7««i» 

I?  ' ; *^'  ' ” , , ^ ..  ^ 

‘ ‘ .'in  f>|'f^'  4-  bo)tp:T/<:0 


1' 


ii  ' ■■  '.*■  . ‘ ‘ . ■”  ■■  1 ' 

K --si't  ..^4A‘T;  flJJ  Xli',  bt^  Xc>  Ufiit  hith  .li  IX^  J'&l.t 

^ ',"■  .,,-  ■;"  ■ ' '*'  ' - ■ -' 

•^♦£vi5i^j»'V-6.‘f.^  £?ritj  OlAif  . 5i^,t.  fil 

v-i-  . • • ,*  ;v. , ■*  ' 

I , A/x*iJ  ^ibikuv  tn>  J5  xa  / .1 

•■'v  • 

i d;i  a 


o-J^r  «;jcr./3cnt>»ticd  .x»W/vfiXiyiiyi;9  ft<i4  i.»v  i'W 


|j  ■; ./?;  ;4  4 *Jlo  iioonA') £i{.•iu^^^ 


i Oito  ftJt  TjOfoXft  '0U4  Ko 

■ r.  ' * /'  ‘ ' A'  ' 

i . ; 


'AM  * 1 iV  P'^l'%  ,W  »A  if  ..*  fi  ’ t '*'V' 


-7- 

cage  and  this  was  partially  covered  hy  a large  flat  "box  turned 
upside  dovm  to  furnish  a shady  retreat.  A stream  of  water  was 
kept  flowing  over  the  floor  and  partially  dammed  to  furnish  a 
small  artificial  pond.  In  this  inclosure  forty  toads  were  left 
during  the  summer  . As  a means  of  feeding  them,  I suspended  an 
electric  light  above  the  cage  and  this  was  turned  on  every  even- 
ing during  the  simomer  to  attract  insects.  A supply  of  angle- 
worms  was  also  dumped  on  the  pile  of  dirt.  In  this  inclosure 
and  without  further  care  the  majority  of  the  toads  lived  thru 
the  summer.  In  the  latter  part  of  September,  thirty  out  of  the 
original  forty  were  still  alive  and  active,  altho  several,  es- 
pecially the  males, were  very  thin.  Efforts  were  made  to  feed 
them  with  insects  caught  in  the  fields  with  an  insect  net,  and 
a specia.1  feeding  device  w§,s  arranged  for  this  purpose,  as  shown 
in  Eig.  1,  Plate  1.  The  insects  v/ere  released  from  the  collect- 
ing jars  inside  the  screen  cage  C,  the  meshes  of  which  were  very 
small.  Then  six  or  eight  toads  were  placed  in  the  cage  and 
allowed  to  feed.  As  the  insects  were  usually  brought  in  late  in 
the  afternoon,  the  light  L was  hung  near  enough  to  aid  the  ani- 
mals in  catching  their  prey  during  the  evening.  The  toads  were 
allowed  to  come  and  go  at  will  from  the  cage  thru  the  dark  passage 
A B.  This  passage  being  kept  dark,  very  few  of  the  insects  es- 
caped thru  it,  most  of  them  being  attracted  to  the  other  end  of 
the  cage  by  the  light.  Two  or  three  mason  jars  full  of  insects 
were  usually  placed  in  the  cage  and  opened  at  once,  and  the  in- 
sects crawling  or  flying  out  of  the  jars  furnished  a continual 


rJS-.rKr: 


TC 


P- 


. ^ ^ - 'P 

^ir 


.f 


. 4 


flu 

^ J^Mfk*JGIi>3 ' ? 


z u 6irtLi>;Vv^j  'A*<;  t. 

h4&'Jff4i  «.  X'V"^*  o^iWijpu 

tn*?X  >»?V«oidr.  «w  nX ' . ttmu 


r^/'"  ■ I?;’ dst  ie  ei^-cm  ca  , 

.Xior*  Wtxf^  " ^ 

-c^^r.*'..-  :«  ''  .oJ^sItra  of  'XsWPfSft^*;’ #>.+:!  r t- 


^ V .-J^i’  1*»  »*tJ"  ;*,0'  ^{^j3ttut>  Cki  o*/i\7  tWDCO.If 

*^*  •'  ''7TlX  llfcijC^  ’iO  »<».a  a^4v^Tt«l  ^V^v. 

-4  Vli  in  U.  t'-  ^•'K  I (\I-  . 1 - . Jifj 

*y<iiy  »v  Xi.fi  XXX^r  1 1*>%  SS^J^t'fo 


1^  “•*'’  .t^.'l^f'f*^  Cfti4  *y;ii\  •>iftiA  flf:!*r  / t*tfi-  •.-+.#/*•♦  rW^ 


' ■ 'Vi 


ii.#n-  « Okfif  n''--*<t|»/..'0  '<;2;,ppc  ;i  JJil, 

r,.  rfvDi^a  ..-i  eXp^^  xf^'iJb^:  .i<i2X£  _«^4  s vut)- ;^.Jt  ;i;^  -.ii 

9 :'■  Tv  '*'■-  ’ ■ 


fit, 


sxy><  iiQ^laW  ’^Q  Oy;i{;m  oif4,  fei.  f«Ai,  ^iji^  j|l 

'•  - , » i!  «!.'•  "^  r 


' :>fU  cnC*  c-l  irS^iiv*  If.  >1® 

ili  ri*\  «f!i2;/  '.-*stf  r,.ib  b.V  Qd!jb^u^ « T’* 


• l^t«|ifc:  ^^).».0:^  »j??  . ’.'^l^l«  >Ya  ^t4  *tiV;A?  r}<U#iVtiJ^X 

» - ^ .i  . 

-5a  ftrtif  Vj  X^T  , -ar I «*•«/* i .ff  A.-w  ' - 

«4d  zhfto  ofi^  oC  'io  Ju«ya.  , :)'l  fii»j£*3^iyi44o^-^ 


no«j«  ootjijj..  a.0  cwt-,  w 

“fi  ;9Uim  /jQ  ®A^  iiPJJaik /iXivttvf.J 

X£4«*>ilP£j  A eiflt  c#  lo  4^0  ^/iX^iXJX  i<3^ 


fc 


^ ,y  “ ‘■^  ^ 

•liVt^ 

gggaV^V.; 

'1^  4l»'^ 


-8- 


supply  of  food  for  the  toads  for  some  time.  Such  a feeding  de- 
vice seemed  to  he  very  successful,  hut  the  supply  of  insects 
ohtainahle  at  that  season  proved  to  he  inadequate  for  the  thirty 
animals  and  not  many  of  them  were  in  good  condition  when  cold 
weather  set  in.  All  died  during  the  early  winter.  Death  may 
have  heen  due  to  the  cold  weather  rather  than  to  the  lack  of 
feeding.  The  toads  had  crawled  under  the  hox  in  the  center  of 
the  cage  and  burrowed  a little  in  the  shallow  earth  there,  hut 
this  furnished  only  a scanty  protection  against  the  severe  cold. 
Furthermore,  the  freezing  of  the  soil  rendered  the  moisture  in 
it  unavailable  to  the  animals  and  this  may  have  heen  a factor. 
Experiments  described  earlier  in  the  paper  have  indicated  how  im- 
portant a factor  in  the  toad's  environment  is  contact  with  moist 
soil  at  least  during  the  early  summer.  Presumably  the  same  prin- 
ciple holds  good  for  the  animal  during  hibernation,  and  a condi- 
tion of  physiological  drought  hrou^t  about  by  cold  weather  and 
lack  of  protection  may  be  fatal. 

III.  The  Water  Content  of  the  Toad 

During  the  spring  of  1921  some  experiments  v/ere  per- 
formed with  a view  to  detemining  the  minimum  water  content 
necessary  to  the  life  of  the  toad.  A number  of  males  were  col- 
lected from  the  breeding  ponds  and  placed  in  cages  in  the  labora- 
tory without  water.  They  were  v/ei^ed  at  intervals  and  the  loss 
in  wei^t  recorded  until  a few  of  them  had  completely  dried  up 


'T'  *1 


r ' 


f * ‘ ^ • • '•  I • 

I '■  I Ml  mi^z 


‘ V'k' 


S .SJIK'  •e^*‘s  «6jio/  tvst*  xol  tivot  *'V  xJL,<y 

, i io  'c*  itro  , ; 4/?«ao6oAi8  rtsT  cM*  Sfrjft 

V ^.’'-  ■ X*  ■-  . ■ (|. 

_ V >4:?  -sMr  0^f‘ 'c6aii6>'S'^T?D 

.-i^'  * • * . '*  '■  ' V . • 

!rt«!i  nciiiiujti.B  ftotv,  ni  titair  tarl/  lo  t.>««  sort  *««  jiXifejiltia 

' ' 

I 


;|jiix.'t  lik  .r.i  Waj 

\ . In  iruoi  -^1  »'  ««»<> 

W.  ' , •',?•'  -^j ' ■■  • ' 

■r  a •taOi-'io  -ji'j  itl  Toa  nni  letetli  Sf/irtit.  liOdojVa.'iy  •.f.-.nJ^ual 

Rt  •'*'*' •rt'  «.■•»•"»  *«XX4*fti  orfj  nl  rl.>j:.i  .1  nsiS 

' ^ ^(TN 

' •■  r ■'  ! 

.'  vn  n&f  0^.7  ’^i’L'.^sr-  ^ \Xr,c^  lf£»;?Qi4i*Si.  i ^ 

jpi  pt^  .^1 .:jcc  To  %4fi,n©ott  o.{^ 

'■".?«Tosa  ^ rs«tf  tuta  eiAiiii-  ^lis,  cp  ?i 

-ii  ««<  i>M.iol6ai  -ijitfaii  o^S  nt  ..t-  hrtfiaattnh  u3r4&..oasS 

*'‘-.  ‘ * '■■  '1  .fli  ^ 


ivUiU  li^hr 


r ^rof-»»f.x>vnti  n'6A<^v  a.l.^  «?. 


-hi'**/  VSTSV.  r,/*  5-'  .V  ' 

'wag  . ’?P 

=-Xi^itta  i).‘*  0’  ,r  -i  ./i  OQiz;;  ■ miiri' 

^Cia  T''-:Vaw-  r4oa  x<^  ;r%jcnfr-  Xm^lsci!oin.\;r^ltf  aoii*;'i 


• I 


^VOT  ©fi:  T o .Tf»^  .?r  o/il'  .rxt  - d 

• '/...  *.14' 


i’j  ^ - 

• * “r 

\ < 


0‘i!>5r  f'.+nei^ixoqpco  oflOR  Xsex  lo  o^i£^ 


‘ '<eJ-dr  scfj  )jp^  w^i bnt-'ol^ 

. ■ ' ' • :"B  ,.  r-  ■'*’-■  ■ 

.-flo;Stv«  JSi,.#.  vM  i«  alt-i  6»t#^o»-.4tXX»S«f  * 


^Vu^Em  ' ^ r 

-stetfjii  bbI  a|?«V,54o  tfUjonjtZg  M.;  ffta  mtrtii  (y»Sn»x'^ 

SMX'  «Jr.-vr*4;<H  fa  b9fistcm  .rp^,  ^itvr:pv  xtar 

««  6*i«-^IajoI®(id.  tc  XlSr,j/  &oi,ooai  J^.'Aw,  ur 

^ .-;  ■;.  1-t  ■■ 


-9- 

and  died.  This  was  first  observed  at  the  end  of  52  hours.  Then 
all  were  transferred  to  aquaria  containing  water  to  a depth  of 
about  an  inch  and  allowed  to  absorb  all  the  water  they  would. 
Several  which  had  become  quite  dry  and  were  beginning  to  stiffen, 
completely  revived  under  this  treatment  and  absorbed  a great  deal 
of  water.  The  changes  in  wei^t  were  recorded  and  percentages  of 
water  lost  and  gained  at  various  points  in  the  experiment  calcu- 
lated. The  results  are  summarized  in  Table  I.  It  will  be  noted 
that  the  average  percentage  of  water  lost  by  those  which  were  not 
killed  by  the  drying  process  was  Z>6.Q%,  while  that  of  the  animals 
which  did  not  survive  was  46.5^.  Those  which  revived  when  given 
access  to  the  water  increased  their  wei^t  by  48.9  gms.  on  an 
average,  practically  doubling  the  average  weight  of  the  survivors 
at  the  end  of  the  drying  process.  (49.9  gms.)  The  average  wei^t 
of  the  dead  animals  was  36.1  gpis. 

The  above  observations  and  experiments  have  emphasized 
the  importance  of  readily  available  moisture  in  the  environment  of 
the  toad,  and  while  the  experimental  data  is  limited,  it  neverthe- 
less seems  to  indicate  that  a toad  can  carry  a relatively  enormous 
amount  of  water  in  its  body.  Perhaps  this  capacity  for  retaining 
moisture  may  be  a factor  in  enabling  the  toad  to  live  on  land 
away  from  the  immediate  vicinity  of  the  ponds.  We  have  seen  that 
in  a comparatively  dry  atmosphere  such  as  would  obtain  in  the 
laboratory  the  animals  lose  moisture  and  may  die  in  the  compara- 
tively short  period  of  52  hours,  (in  one  experiment  five  toads 
died  in  41  hours).  This  fact  may  perhaps  be  correlated  with  the 


. n Ti  or:.  I*;  0.-9  ftSJf  «idT  .'fiOt^-fjb^, 

' Sv  ■“  '■  fc.  ■ 

■ . , ■ ■ *,Y'.  - 

4rfc»‘er  v„*  iv*  oi^  jSrwoxXr^t^  rtt)r,r 

’ • , • '^  *< '-  , *3  " 

nlrU,-xis4>w 

l^i»  «*'*.. ij- cj!9S-*:c»^*j_  &£|jjt  'vtf'ff  1;/j 

, --.rjJlHr  o,::  r.i  t\i6:i  ptfolyt,  Ju  hKtti.4^:^  bii.'' 

-^tf  *X^w  '■  -V  If/ 

t^i>^  rirti^vr  V./  ^*oX  tfe.‘,,*r  :p  f v(W 

fik.£^iu«  tfdf  to  o-.-i.'*  \ »«'  ?^t».:^*x^  yrl^^?C.a^’• 

.y^tt'ivlV^  zSf.--.w  b->rr{:^  dot:fw  ijnc.VP  .•>-.'  v.-m  ofX>?i-a' Jpu  yic  ‘^X4^ 

ac  «r0  .!>;;;* . . . ^ ^{C|  cii^UJaSitt*. 

<i-:9viir.-.K5  9r  J £ rp.^-v* >m?  ,,.ai.fj,o-  -{Us^i.-Wii.  .‘(r^jj-tteii' 

^itviLT*)  9^x*y*  “iC  ’.viy;  S.Vfj,:  .’tn.trir  sraxW  '”1  ' 

. e-^V'ri-sntjxo'  b.ia  r ’ 

Ic  t>v^  m 5i»  :>ii4£«y  .^o 

-r.*.y  *wr«fr  XaJ4iL>!!::i-lw<iKi>  qX'^*'.JT' 


,yj*o  tfoj. ‘ii  Xh'ul  j4  :,  HI.  aii^-edi 


I:s»3k/oo  sX^  fli' ^3.^4j ’^'ix  .Vi?^vV.i^ 

<^#:^^no  sTi-t  c.^  ^niJ;ci>^9  ni  !Xcc.&«V‘t  «tf’ 

67A4^or;'  .a2.«o<f  orfj  to  x.^-iraoi:tr  o ;j  ^ri  x/imf 

-£-2i5^qa^  4i':  oi,V  v^..^  »XAnd-*iU...<»^^^ 

^ *ir..^r>i  |V'n  t.o'  rf-xojfe 


-10- 

reactions  of  the  animal  to  gradients  of  evaporating  power  of  the 
air  as  shown  by  Shelford  (1914)  and  indicates  that  these  reac- 
tions tend  to  keep  the  animals  in  their  optimum  environment  in 
moist  situations. 

The  question  as  to  v/here  in  the  toad's  body  the  water 
is  taken  in  and  where  it  is  retained  has  not  been  answered  satis- 
factorily. I have  never  seen  a toad  drink  in  the  ordinary  sense 
of  the  term  and  it  seems  unlikely  that  any  appreciable  amount  of 
liquid  is  taken  in  in  this  way.  When  a toad  which  has  absorbed 
a large  amount  of  water  is  handled  roughly  for  a few  minutes, 
much  of  the  water  is  expelled  thru  the  anus  and  possibly  this 
might  be  where  the  water  is  taken  in.  A more  likely  place,  how- 
ever, would  seem  to  be  thru  the  skin  by  some  sort  of  osmotic 
action.  Possibly  it  is  stored  in  the  lympth  spaces  beneath  the 
skin  and  passes  from  here  thru  the  wall  of  the  bladder,  when  the 
animal  is  disturbed,  or  it  may  be  stored  directly  in  the  bladder. 
These  points  have  not  yet  been  studied.  Some  preliminary  work 
has  been  done  on  the  skin. 

IV.  The  Absorptive  Force  of  the  Toad's  Skin 

Reid  in  1890  studied  the  osmotic  action  of  the  skin  of 
the  frog  and  reached  the  following  conclusions: 

1.  The  normal  direction  of  easiest  osmotic  transference 
of  fluid  thru  the  living  skin  of  the  frog  is  in  the  direction 
from  the  outer  toward  the  inner  surface. 


,t*  -u  ix:rr^.;r^  JLi.rl.tn  ejt;  .1,  f 

■"V*  ■/: 


f • f ^ ■> 

1#^  fli  stA6«\/io-£vriD‘iipTL'jij?^ 

■*  ' • »',.*  r ' > ' ■ .a  ^ •■  ■ ■ • 

^1  ^ « 


^ 'V  ^ 

Xj.  ' \:  , 

. ; 7 . ■ ; ■ ‘ • ''tiJiuEta  j'^i'cfl 

I ‘#<*  «>«  r:-i  ,it  t^\xat3*rL  ,rtn  ■ t. 

t w * i • ' 

- - '»'V  Mi*»3  riifr'f 


M -«  «i.  jliiStJ.  »,  »iiriiir  i ft 

.. V ^ . ,.  ’ / .,  , " 4t;  1 ; 


ii  ^ rt,ur  .^nr  ciS..  til  ri 

j[  r . vo‘ :ii4v  -^^.ws  -x?/;  xA- -'  ni  ^ ^ux 


axx*cat»9  -iv-  r t.'K  -^..wioa  ;t;,>.x  ^,tj  tor^ 

''■"  ■ "'al^ 


r-i.  J ifw  n<:i  n -ki-C  ' kj 


r 


J ,lnr.A^  O -.J  lo  /lev  f>  w*  irrx^f  ^>Ttva 

.7st  ijsf  il  e^.1  >i.«»*-tio  i*»t«3(  W v«  ri  TB  .ftwru/Aifey.!  i«*t«ii^!;  i 

,-  j{%ov  Y-z  ititrx^et<f  s/ioo  .b^iitit«  a.-,.  1\  i ' 


Ji%ov  'cz  ititrxfet<f  s/^oo  .iid,tl;xij«  iivsi-  ^eu  ®v-0rf.  o^'oWdtP 

f.  “■  - i f ' 

if  , I xT..  ^ .„_  ,X  . ' . , 1 


'Y  r I 


' r - .j 

iil^U  i4*^6(ZT  8)^,?  ><y  .V*X 


j_  >-r  ^ 


P ,6  y<f$X  ul  ^idg  . ^*4 

'*•  i”  ' 'V  ^ ■ X F ■ '■  t * 1^  i'  ^ 

, ,.  ,j:cuxi*sui3..<^  »n>«f(«io;  #i»  ;6orit.4»:i  fin«  *ortY  siU? 

Oii.o»)i<r  io  a»>4'ocjti6  .ii-ji^or,  ,£*  '^. 


■ . (»iv'o«-io  r4  »jf  g„t  ,ftf  •*«  ^-,iX  »(U  «TU«  xJ. 

Ijfc  * ,1*  \ ^ • V •'  j '^*  ^ 

• t,  / ^ ^ 


r-a-art.T 


^.oos'niid  eit?  jbiiiw  »£4  xjwi' 

.*do_  __S'S;  ■•  ...  . 


-11- 


2.  The  transfer  of  fluid  in  the  above  direction  is 
intimately  associated  with  the  condition  of  the  tissues.  Condi- 
tions or  agents  tending  to  depress  vitality  diminish  the  transfer 
in  the  normal  direction  whereas  stimulants  tend  to  augment  it. 

Reid  suggests  the  possibility  of  a definite  absorptive 
force  exercised  in  the  living  skin  which  mi^t  explain  some  of 
his  results  and  he  records  three  experiments  in  which  his  dis- 
charging osmometers  showed  a definite  action  taking  place  thru 
the  skin  when  the  same  solution  was  placed  on  both  sides  of  the 
skin,  thus  eliminating  the  possibility  of  osmotic  action  in  the 
ordinary  sense  of  the  term. 

It  seemed  worth  while  to  determine  whether  these  same 
principles  applied  to  the  skin  of  the  toad  with  its  more  terres- 
trial mode  of  life  than  that  of  the  frog.  For  this  purpose,  I 
devised  a simple  type  of  osmometer  shown  in  Fig.  3,  Plate  1. 

An  ordinary  Harvard  staining  Jar  A,  is  provided  v;ith  a two-hole 
rubber  stopper  B,  in  which  are  inserted  two  glass  tubes,  C and  D. 
The  lower  end  of  tube  C is  flush  with  the  lower  side  of  the 
stopper,  while  tube  D extends  about  inch  below.  To  prepare 
the  apparatus  for  use,  stopper  and  tubes  are  removed  and  a small 
piece  of  skin,E,  is  placed  over  the  longer  tube  (the  inside  sur- 
face of  the  skin  being  placed  next  to  the  glass  for  example). 

The  skin  is  drawn  ti^t  and  sealed  in  place  with  sealing  wax  F. 
The  Jar  is  now  filled  level  full  with  the  solution  for  the  ex- 
periment and  the  stopper  is  inserted  in  place.  This  forces  some 
of  the  liquid  up  into  tube  C,  and  if  the  apparatus  is  properly 




(px  - -iapitE-jr 


•XT- 


W . i>  t '^c,  «0X>^4^  «.*iw  " 

>'^3*^.1.*  P.;^  rtni,aa^ 

.^i  o«  n4n»£ijpXJ-r;  sAM^0‘i  ‘ 

f.'m.  ;u.-.;^-4  :.*%i«  ,f'.“.tir  'Ctisuf  jairi:  oa:  r»i 
-<Ei>  bXJ;<  .fcrXa^  >i  ?t^Yrf:?‘ OA*:  r^o-i  f»xt  A-ri  crfirKiK^  stj>  ‘ 

.,4^^  «,tx^niit  m feci^f 

; 0/U  -Ja  «p  ^14-^9X^  -.;r  ooijui^p  *>jcf 


••■■•.f-ra 

^/.{T  jX^  i\tif  >0  IftSOr-ft ->,Jj^Jfc;U-i>.tO 


« • • ' • « ^ ' 
o-liM  «»;’,?'•  rt^^^^t*)4ji»  or/X'Jwr.'-i.^  cit)  ^ X r**:v  ' sH*en»Mi  i,I.4^jV 

_ . ^ 4 . = ,"  ^ ^ ■ . ' *v  J 


viXOC!  0^4  *>.V  Vior  IX.  .'^i^;  ..rfi-  CX 


'«r- 


i ,^yi>rrir.f:  oW  e-'  t.  ,&,vS  k^j. 


>l  v:Am  ^ n»w*ia  .H»rC>?  AX‘!;r'4'j?’>||Aftl>^veA 

« ttJi#'  V»lv;v0-».!r  srX  ,i  ^*t.;rTL«K  ttk-^ 

- ente  W 'lAliir.  /U.  iti-dJui 


la  otU»  5^>rr:  ^fiX  rOi^  xX/Kwri  .,jjb  ? aJraX  ■ 


y‘,^  vT  . fCXpd  i(6<a  i a*.cc^  »>i*f0^ac6  eifj^ 


XXii^^^  o-j  , *^^vlv  »{i.  , 0i»y  icl MX 


-T<#tl  ©bXc.-il  04iX;^  rcfi  ‘.WP  m ’C  -fb© 

if>4w  flX  i^X^e^br44ia(%ij  wjaTb  tX-;?i5t«  wi-X"; 


-tsatff  riea'  amutfip- ai/*  lO  iv-^iXi^^  jr»7!»i  .hsxiJtt  woa  .V  '^  >>  •>_.«} 


^ e^t^j  9f-/5?  .#arf<(.,  ni-  if*<i»sni  «i  ^vsfWo'aSJ  A(u«.:«|»ia««qj 

■■  ^■^--  :h;.^  v:'Kt-  ■■■•  ■ •■">" ^.-  111 

\ ./ 'rMt  ' ■.'■0&S.I  ■ ' . jJ'.'V.'"’"'  '"•^'•  iJ  “-'■ 


It'.  HLt 


<-■? 


-12- 

made,  no  air  "bublDles  will  remain  in  the  jar.  The  same  liquid 
or  one  of  different  density  is  now  poured  into  the  top  of  tube  D 
to  a convenient  hei^t  and  the  levels  of  the  two  colimms  of 
liquid  adjusted  until  they  are  the  same.  The  apparatus  is  now 
ready  for  use.  It  is  set  aside  and  readings  are  taken  every  few 
hours  by  measuring  and  recording  the  heights  of  the  two  columns 
of  liquid.  If  osmotic  action  takes  place  thru  the  skin  from  the 
outside  to  the  inside,  the  liquid  in  tube  D will  rise  and  that 
in  tube  C will  fall;  if  from  the  inside  of  the  skin  toward  the 
outer  surface  the  level  of  liquid  in  tube  C will  rise  and  that 
in  D will  fall.  Thus  we  have  a means  of  measuring  both  the  direc- 
tion and  the  speed  of  any  osmotic  or  other  action  which  may  take 
place. 

The  tubes  used  in  these  experiments  had  an  inside  bore 
of  7 mm.  and  were  of  uniform  size.  Each  tube  was  flamed  before 
using  to  remove  any  trace  of  grease  which  mi^t  affect  the  cap- 
illary action.  Tubes  having  an  inside  bore  of  about  7 mm.  were 
found  to  be  most  convenient  for  several  reasons.  If  smaller 
ones  were  used,  it  was  found  to  be  difficult  to  pour  the  liquid 
down  into  them  in  adjusting  the  level.  With  a tube  as  large  as 
7 ram.  in  diameter,  a pipette  may  be  thrust  down  into  it  and  the 
level  of  the  liquid  adjusted  by  means  of  this.  On  the  other 
hand,  if  a large-ended  thistle  tube  is  used,  the  piece  of  skin 
becomes  limp  and  stretches  and  sags  in  such  a way  as  to  make  the 
results  inaccurate. 


• ‘ail  I ^ 


■ V“' 

* j ’ ' 


. , ■ ; . . — ;-i 

j|  < io  ^.NJ  ‘O^c^x  Ai>^c;o<5 Vrni  fciz-V'i»c^ 

«,'*  /''•'•■ '*3  - cr?»  9#sy  .vx««  eaj’^^*  tt^M  bnsi^fy,j  >iiuhh  f 

' ■.  ' ■ » '.>  . ■ ' ■ V 

‘ n *V  A.tr  r>  ...  J — A i.  . . ' . ‘ ^ 


T-*-  V’xi^vn  rj.if^  ->t->  © Jf.A  ,x  it 


.t>«v 


_i*  _ Yu*  .•<  crwt.'  ^)r»^  5.:fJ-u/|w^r  ’.•:<(  »nv^K. 

'.  rar-i . 'tt'utr  it  .'iifnii  *^0 


»■«.•■-  Jjc,:  ft  MV)  rri  i**WW  •£»  . •<»  OJ 

oj;.*>f!'r^  al\-  ‘i’i  '^m-laaxx. 7!  iJU«5  JtWw .0 *<)«  nl 

■ \-'iii&  lirt..  f>e;-i  lllw.s  f^ivt  Hi  i/Af>|,i;p,>6  Cfty^jT  f;i,'  e^>;4i7{i^»«/o 

♦ 6 . . * 


,,  .aXjkVXii^.  a.ni 


V'  ’ ir  *, 


Iv 


F 

o^nXc 


V 


J?) » 


r.'  ,ii- 


aicdd  flii  ni‘  S in'Tj 

■ a-iCiirf  »MKX^«Vr;iAi»  doia  .txia  mMim  .lu  ww  •Ma  ^«r  7 to' 
...o  pi  , ini.  ttu' iii^ljlr  .•f.iii'tw,  enj«--i^  it?  »3»it;J  ’^(le  PVPiUftS.  oi"^5^Hiai/. 
aio«  .etx  V iu-'a-ioft  piJoj,/  iv  atliy^d '«ajj/(i  /. 8*1*4  t*»Ul 

TI  ■ ;».■.. fc«t*ir*i  -...-  l*ia-S»»i«*;i«M,  i.£'  or  teftol 
=.11..  ,j«,  „j  ftX^^IXipi  t,  P^-OJ,  Jirw.ot  ..ov  M MOo'^OHa:',': 

1'  aJ!.;  i-.w.JJ  .oiftf  .aolor^Jt±,,;fi;|.  ^ T-j!| 

I t6!Uc  9(U  n«  ^ ^.«iir  :o  a,K^  Vi  la^sut^'t  iiti'eti  a.-a  -^o,  JaraX-^J 

| : lU^  i«  0oO-lj,-0  otful^oiloX/W  6p),n«,-iBiyil 

Ijf  1^  *.  ‘ * * 


vji04ii*,i  fj  Xl,*acii  j 

- ' -ll^  c - ^ 


1-1  1 » \i‘i 


-13- 

Results  with  this  type  of  osmometer  were  recorded  on 
the  experiment  sheets  as  shown  in  Table  II.  Two  columns  were 
arranged  for  each  osmometer,  one  marked  (Column  4)  referring 
to  the  tube  which  was  closed  by  the  piece  of  skin,  the  other 
marked  - (Column  5)  for  the  open  tube.  The  heights  in  milli- 
meters of  the  two  columns  of  liquid  were  recorded  at  intervals 
in  their  respective  columns.  The  difference  between  the  two 
columns  between  each  two  successive  readings  was  then  computed 
(Column  6)  and  the  number  of  hours  elapsed  is  calculated  (Col- 
umn 3).  Results  may  be  recorded  in  tabular  form,  showing  the 
maximum  difference  between  the  two  columns  during  the  course  of 
the  experiment,  as  in  Table  III.  This,  however,  does  not  show 
the  rate  of  action  taking  place  at  various  times  during  the  ex- 
periment. This  may  be  shown  by  plotting  the  results  on  coordin- 
ate paper,  representing  on  the  ordinate  the  number  of  millimeters 
by  which  the  hei^t  of  the  positive  column  exceeds  that  of  the 
negative  column  of  liquid  and  on  the  abscissa  the  time  in  hours. 
Graphs  I to  IX  illustrate  this  method  of  recording  results. 

Table  III  and  Graphs  I to  IX  give  a summary  of  the  experiments 
which  have  been  performed  to  date. 

In  the  experiment  represented  by  Graph  I,  the  main 
reservoir  of  the  osmometer  and  the  negative  tube  were  filled  with 
distilled  water,  and  the  positive  tube  was  filled  v/ith  a solution 
of  higher  osmotic  pressure  (5^  cane  sugar  in  distilled  water). 

Two  osmometers  were  used,  one  represented  by  curve  x on  the  graph, 
being  arranged  with  the  skin  placed  in  the  apparatus  with  the 


,trT4T 

' V''  *'*■ 


-IT-W  •.O.  aeroo  attuKotl 

O.t'V  n,1Wi/.lrKl*T  .n  sxi:Hi<I  «/■  r*c.-fe 

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(».  bex-jw,  1 

...  ■ '?h 

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1;,.  «t,3-...3,„-<s„  «jWc6  "I 

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1v 


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r MfJ'H  : .mA  0“' r.,w>v..4c,it  w ^..3  e-Xujf^  . 

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.■P*)  '^^>r  Di.Ni-.vC99,  oiB  ,tfX,  J.r/ 


■'iii 


V >! 


M 


-14- 

outer  surface  down;  the  other  represented  by  curve  y arranged 
with  the  inside  surface  of  the  skin  down,  thus  placing  the  li- 
quid of  greatest  density  on  opposite  sides  of  tlie  membrane  in 
the  two  parts  of  the  experiment.  In  other  words  curve  x repre- 
sents results  with  the  cane  sugar  solution  of  the  inside  surface 
of  the  skin,  curve  y with  the  sugar  solution  on  the  outside 
surface  of  the  skin.  A comparison  of  the  two  curves  shows  imme- 
diately that  the  osmotic  action  took  place  readily  when  the  sugar 
solution  was  on  the  inside  of  the  skin  but  not  at  all  when  the 
solution  of  greatest  density  was  on  the  outside  of  the  skin  until 
late  in  the  experiment,  when  the  action  ceased  in  curve  x and 
began  in  curve  y.  If  we  assume  that  the  tissues  of  the  skin  had 
died  at  this  point,  then  the  experiment  would  seem  to  corroborate 
Reid's  conclusion  regarding  the  frog’s  skin,  namely  that  the 
normal  direction  of  easiest  oxraotic  flow  in  the  living  skin  is 
from  the  outside  toward  the  inside. 

Graph  II  illustrates  a similar  experiment  in  which  the 
fluid  of  greater  density  was  some  water  expelled  from  the  bladder 
by  toads  when  handled  rou^ly  as  above  described.  Presumably 
this  liquid  was  of  a greater  density  than  distilled  water,  altho 
this  has  not  been  proven.  The  general  trend  of  the  curves  is 
similar  to  that  of  Graph  I,  but  since  nothing  is  known  of  the 
nature  of  the  liquid  used,  no  definite  conclusions  can  be  drawn 
from  the  experiment  at  the  present  time. 

A number  of  experiments  were  performed  using  the  same 
liquid  on  both  sides  of  the  membrane.  Three  liquids  were  used 


» * *r 


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r ^ _ , . . . ,-  . 


t'^?,i:;«  f>  1»1  «:  jp  >/i 


i%r.j 


'E^ip,*'4rib 


4 ^ ^ur.  V..  . V-.,  <-  !<•  r;<y  »)ar 


it*  Tr' i M 


*’«  »■»-«# 


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'j  x pr^u.t  fiC®  o,v  -«•  * 

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™ ' *X  4 * 4Tfo  iji 

4 >yr  ‘ '•'' 


ry*f.  s '^^'€^4#  ft  frill  ’...i,^  n"^tvA  tpk* 

t ' ■ ^ 4i '5,0'' . or*  >r;i0’x**^e’2  hcts'xit'tlqk 


I e.*?  ftf  v.,n  micAt^iu 

' .*  ' 7 ;*‘|l 


, if"/- t ^cvC'# 


i 

.1  © iJ  r.i:  «^iia»:'rt4'g:p  *?  ^ je:s.t^^i’XI  UI  rl/A4i'cO  ■.. 

is;>!>arrf  er-f-i-nn^  V>r:el.-;a  m*aW,  »*r  V^itra.©  tpJjjs,,'  1& 


tvoiir-g^  3>t^rinMtt  ggesi  at  uoS x^ 


f3^:f^'  " , »xiJ .'  ■>/  5 eXr  I ^ f^'  -.t 


Kjr  ns-r-tuo  ^’'■^Skk  J 

f>r'fj^\<.  pj  ,ia-j,on  to  i«ctr' 

■--■  -*■  .'.i.-  . . ,.  ! - Iffl  * > _ , -4, 

^4'^-' l^5*T<{  f>i4r  >)  t0AtjLto?rt?) 


-j 


S'"  ' , 

* •*-y^.,?:>»  S.4w  »i<w  t*  ifl^. 


irrb^ei:  jf^.^  i»©<l^^pXX 


Pf  ■ 


-15- 

as  follows: 

1.  0.6^  Nad  (Approximately  isotonic). 

2.  5. alcohol  (as  a stimulant). 

3.  A weak  solution  of  chloroform  in  distilled  water  (as  a 
depressant) . 

In  the  earlier  experiments  of  this  kind,  the  skin  was 
removed  from  various  parts  of  the  animal,  sometimes  from  the 
hack,  sometimes  from  the  helly  or  from  the  side  or  the  breast. 
Results  seemed  to  indicate  that  a decided  flow  of  liquid  took 
place  thru  the  skin  from  the  outside  to  the  inside  and  that  the 
strength  of  this  action  varied  with  the  solutions  used  and  with 
the  condition  of  the  animal  at  the  time  when  the  skin  was  re- 
moved. Later  experiments  in  which  tiie  skin  was  always  removed 
from  the  belly  side  gave  different  results,  showing  practically 
no  action  except  in  the  case  of  the  chloroform  solution.  These 
experiments  are  summarized  in  Table  III,  Nos.  2 - 6.  A further 
study  of  the  earlier  data  suggested  that  the  absorptive  force 
might  vary  according  to  the  source  of  the  skin  on  the  animal’ s 
body.  To  test  this  point,  Experiment  8 was  set  up,  using  skin 
taken  from  the  back  of  the  animal.  The  results  proved  to  be 
quite  different,  as  is  evident  when  Graphs  III  and  IV  are  com- 
pared. In  these  two  experiments,  conditions  were  exactly  the 
same  so  far  as  known,  except  that  the  skin  used  in  Graph  IV 
was  taken  from  the  back  of  the  animal,  while  that  in  III  was 
taken  from  the  belly  side.  Apparently  quite  a difference  ex- 
ists in  the  absorptive  force  of  skin  taken  from  these  two  regions 


Vi»ir  c;<  ©i»i  ) X .£^:^ - . i< 

•■^  ■ ^ , 


'i  <.T  .- 


: ti 


n* 


. . .r’  , „ 

■ ' ’ ’■’  ■■  ''  ‘-.'*13'-  ^ '. 


.....  /.■»■•■%• 
^ A V..,  - . 


i.i.ij  ,5/ii.i  bi -J*  ‘Xf  f<  "t  ->  -x  ► ,[ ’.i  . n O'fi  n;X' 


aAivj^oinoa  .i^^arlr*  {au^.^*,  biv'^ 

'■*  *.  ^ 

.tnm^  0-rv*  aa  oMe  ^-.:  tUiKf  - 

1^,  cit.5  -*nrfo  jif?r  n/;?  ■ &a  /jr-;*.  j,c*a  /a->:c  r 

;,  lift-  ^ M’.t  Hal  f.  fit  f -V  It  .;b!-iitt  n<>jja«  oijU  ’lA  ^{^as^l■•1i. » 


»rt;  *.‘J»‘'i:-.t  .is  .Kri  tt  k;i  1o-j(»A/tCb<.;  »j{# 


“ _ r>pn^9'i.  B.>.  (•4>;n.o»i>-^^ffl. 

iitiropa  .■.Jij.-ao'i  rf!r.ji'..-il:a»  »"i;i  «iJi>  v;  £«</.,,», >ij!  *iv-t'l 


.nioir^y/cki  «i^*j rA<v.ii<i>  «.-}  2: o o •#<*;> -M  aj:  ^ Qf»  ,". 
A .5  - fX-  ..io,^  .Alt  o:,rf«T /j 


%4»JtXii5o  #• 

^ vii^  nt  -rl:lt  W t»o*»aoP^^ 

r-tUj  . U(  V^/lPWirzaq;^  ^ 

.-/tod  XXI 


‘I  '. 


• i 


ri 


VI  ri  ni^Si  , tV^«fA3l*  |tr.  mi 

i>nr>  +*w-s  'r__.  .... 


ovjir  in  Hi  .tAttf  ©liHv  ,i.mtsJc.ii  ic  >idijjcf  axij  «%[^^! 


A 


,J»  '■  ‘ :'  •'*  ' .->/*fi'r  ■ 


■ .^i- 


-16- 

of  the  body,  the  action  being  strongest  thru  the  back  skin.  This 
fact  is  also  shown  by  later  work.  Possibly  this  difference  may 
be  correlated  with  differences  in  the  structure  of  the  skin,  the 
presence  of  poison  glands  on  the  dorsal  surface  or  some  other 
factor  not  yet  studied. 

Graphs  V,  VI,  and  VII  show  the  results  of  an  earlier 
experiment  in  which  the  skins  of  three  toads  were  compared  as 
follows:  Skin  No.  V was  taken  from  a normal  toad.  No.  VII  was 

taken  from  a toad  which  had  been  allowed  to  dry  up  and  die,  and 
skin  III  was  taken  from  a partially  dried  specimen,  representing 
roughly  an  intermediate  stage  between  the  other  two.  Results 
indicated  that  the  absorptive  force  varied  markedly  with  the  con- 
dition of  the  animal  when  killed.  Unfortunately  however,  the 
skin  for  these  experiments  was  not  taken  uniformly  from  the  same 
part  of  the  body,  that  for  the  alcohol  and  chloroform  being  de- 
rived from  the  back  and  that  for  the  Nad  from  the  side.  Hence, 
in  the  li^t  of  the  later  work  indicating  that  the  skin  from 
different  sources  differs  in  its  absorptive  force,  this  experi- 
ment needs  careful  corroboration. 

This  was  begun  in  experiment  9,  Table  III,  in  which  a 
partially  dried  toad  was  used,  one  which  had  lost  31  gms.  in 
wei^.t.  Skin  was  removed  from  the  belly  side  and  from  the  back 
and  the  two  compared.  Results  were  quite  striking  as  shown  by 
Graphs  VIII  and  IX,  chloroform  accelerating  the  action  when  skin 
from  the  belly  was  used,  whereas  alcohol  produced  a strikingly 
similar  result  in  the  case  of  the  back  skin.  What  the  signifi- 


■>» 


,1 


r 1 * * 

r.x/Jt':  if...  ■>Y^  inf4  :r^4>^^ciicc  aoWoi  ©i</  .V 


r ‘.0  0‘uCwiJi#  '»,.j  xU.  ei»fiJrf•l:^^t  “ ' 

QMV**  'Xrt  pojr^.-:uf,  i.  tt\gj),  r>iW  ,|h;  (Juc'it>^ 


# ■ ' * '•s  ^ 

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’•  ’ < ' r " V,  * 

‘iv  otiifi)lj,f\ta 

^ \&  f^J»uyQ-t  #a-  av.x>r.^  ,r>/::*  V 


r.-.  Ct6i  ioSiVa^,.  t.6td,  „t 

m..  •**•1^  ^ -1  ^ . V»  . '-  -' 


^cr  .; » V Ji..:2fh  H-crorl  f!0X«4  urv  7 ' 


L-Uf;  ' .6;'.  N^.p-Tti  <>J'  ^‘■'■^^;^^/.'t<-~^‘<.^.i  .-aicV 


- nif  D-a  \xie.o:.^  -Xiri't-v  ftOTJU-t  f»vi.*  ,%oti./ii,o/;i  7ni^^^4S^il),Ti  • | 

^ ,..  'XT-'ffW'  .3f*;ri,y 

Oii&\XiO-r^  vXh^miflb  h itln  A-:^:  » > • ....  » ^ 


4LCA  ^vV  ;x5^i4 

I'  i«r:oC.Xr  b/<J  t 'A  J«!i  ■ » _V^  Jb||s’X^ 

mW  ifei.M  • f,-,.t'Vi  •»  -.-.1  9...  -.^  *'  •?'■ 


,,fO;.6R-  ......  i,-  m S^-.  t,  Xuijx  «;'‘.r>ta  * ^rJT  .-A,.,  BCTi-J  ■„, 

.ClO’tl  Iti‘>*'i  .nftif  .tcs-’4*  Ifc-  Ik,...  ....  ...  '2 


■rxo'tl  ^iU  lt3(.  X?|n 


9Xi:5.  d®*  r:^ 

• ’>raB  fe 

X " ’■  ' .*  , » ' '♦  ‘ Olf ' Ztj'X^'l  Jjf)  e/^ 

■i  rtdir,x  ‘TX  V a«w  J 

3tr>aX  D;t  TC^7'X:  SikvxSigtex  cj^ 

X pi.mr,  J P*»^^  iSv 

..  ai..^xo^itv  0Oi^oM.,.0ix^  hJiX^^'nilooof  m-Q^qpXKn  . .xtbu^  IfXJ^ 

'1^,  "■■•i*;  ■ _ ^ ^'.  ..,  *'  *■ ':  --*■•  *^>.  * .i'-‘j’‘  ''.j  . • 

■^--— „ V.  ,,"^,  ;'j}^. 

':«■■  - _-,.''liii._.  ....  ^ VJ-  . 


,t- 


V 


UB 


-17- 

cance  of  this  may  cannot  toe  stated  at  the  present  time,  tout 
in  this  connection  it  7^ill  toe  noted  that  in  experiments  2 to  S. 

(Tatole  III),  where  skin  from  the  toelly  was  used,  chloroform  was 
the  only  solution  which  showed  siny  noteworthy  results. 

As  regards  skin  from  a normal  toad  in  contrast  to  that 
of  one  partially  dried,  Graphs  IV  and  IX  may  toe  compared  and 
show  striking  differences  in  the  results  in  the  different  solu- 
tions. 

Conclusions 

The  data  so  far  gathered  cannot  toe  considered  as  suffi- 
cient to  warrant  the  drawing  of  any  definite  conclusions.  It  is 
merely  suggestive.  As  mentioned  atoove,  Reid  ( 1890)  as  a result 
of  his  work  on  the  frog’s  skin,  suggested  the  possitoility  that 
some  positive  atosorptive  function  mi^t  toe  exercised  toy  the  liv- 
ing cells  of  the  skin  which  would  explain  his  results.  In  other 
words,  he  put  forward  the  hypothesis  that  the  living  skin  exer- 
cised some  force  comparatole  to  a force  pump  and  th^t  this  force 
ceased  with  the  death  of  the  tissues.  This  seems  so  striking 
an  assumption  that  very  careful  work  must  toe  done  and  a great 
deal  of  data  gathered  toefore  it  can  toe  considered  as  proven.  I 
merely  wish  to  call  attention  at  this  point  to  certain  aspects 
of  my  experiments  so  far  that  seem  to  fit  into  his  theory.  In 
Graph  I as  toefore  mentioned,  the  osmotic  action  seems  to  go 
readily  from  the  outside  surface  of  the  skin  to  the  inside,  tout 


l>uava.yta  stf  \9  'Isihjo  | 


^ ^ ^ W •-  -V  '*.'**^v  11 

.s  rj  t?  <tl  J 


i/^iU  O^-  ni  n ‘jsx’ir,  ai  iii  •.; 

■if»  f*  * ^'S.-' T'  ’ 


Ff  > 


«■  'biu  A»-^c=c-.  f-;  ^:,  A'fr  - V'l  ,<•  .^C 

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_ .a'  '■'-  ■■■*' 


.. vv;:'^  tti 

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/ -; 


f^ ' 


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';t 

>1  » . 


*i 


W- oO  ^ ^ 


»•’■;•' tyi 


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a f 


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^:.  . 'VVW  .A  .*'ij4r^ 

..$  ^ .'f;.:i^-^ 

^' •■*■'•*  ^ i»p»iri'«»-  M i^taj  •««{:#», w-:  9»'iw<'» ^ 

•Ji/.'a  f.l  .;*>U.«5S;t,aUJ  iTi/a:j;j.f,  (ioigs,  ^.-  'J 

'--«Ka  -a-.:  J..avu  «.;,V  «{"*«/,»  av.^  0^.  ^ j,,,"-/-  ^VoW  '''" 

• .,  1 ■•,  , •■i  * * ,7/^ 

nJ/41  tk  .r I *•*  ’- 


?nto:iw-5  ‘ . .io^  'o-i,  ) 

1®*  - T'"'  « t^Vcy.'jr'i  jIc.  **  ifKk.  w*  ^ . 'k.  ._  : *t’ 


r-  ncVfli^tBV  sA  (WO  (fi  B'to'tB-  *(.-«., '.'J,a 


rJ  tiler  SIX  VI 

-*  • , ‘ * , • . • -*.  d 


4r 


. t.  s^-,  i.wpe  :.foSM?HJ(^.«»  ftioi»rf  „-.r  , ?^«| 

...  : ■ . . J,  ..  •:  - . V « ■ fA 

fS.  - ^ 


>*^  .3>ii9«  .^,0?  ,?«|»  4l^e4tf».»V'  «#V  ' 


•% 


-18- 

not  at  all  from  the  inside  surface  toward  the  outer  one  until 
late  in  the  experiment.  At  approximately  the  point  where  the 
curve  X ceases  to  rise,  the  curve  y "begins  to  rise.  This  would 
be  quite  in  keeping  with  a theory  that  the  tissues  had  died  at 
this  point  and  the  absorptive  force  exerted  by  them  had  then 
ceased.  Hence  curve  x ceased  to  rise.  The  skin  represented  by 
curve  y also  having  died  at  about  the  same  time  now  acted  as 
any  non-living  membrane  and  osmotic  action  took  place  toward  the 
liquid  of  greatest  density.  V?hy  curve  x should  fall  to  nearly 
zero  is  not  so  readily  explainable,  but  bubbles  in  the  liquid 
confused  the  readings  in  this  part  of  the  experiment  and  hence 
perhaps  the  curve  should  not  have  gone  so  low.  i>lo  bubbles  formed 
in  the  other  half  of  the  experiment  to  confuse  results. 

Another  point  worthy  of  note  is  illustrated  in  Table 
II.  At  the  bottom  of  the  table  is  a transverse  column  headed 
^ increase derived  as  follows.  If  we  glance  down  Column  4,  for 
example,  we  note  that  this  column  actually  rose  only  one  milli- 
meter in  hei^t,  and  a number  of  other  columns  did  not  rise  at 
all.  In  the  chloroform  part  of  the  experiment,  the  positive 
column  rose  2.5  mm.  The  difference  in  hei^t  of  the  two  columns 
was  due  then  in  a number  of  cases  to  a lowering  of  the  column 
in  the  negative  tube,  not  to  a rise  in  the  positive  side.  At 
first  thought  this  would  seem  to  indicate  that  no  ’’absorptive 
force’’  had  operated,  that  the  difference  between  the  two  columns 
v/as  due  perhaps  to  evaporation  in  the  negative  tube.  The  sli^t 
rise  in  the  positive  column  mi^t  have  been  due  to  temperature 


I wrf  T ,j 

TTa  -B/*  . 


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-n  ji.  rM.'  al,  „>>  %-^tfb;.. 

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•tc.  m rx-  JOfi  b4i>,.l)ficV/Xt»tr,  Itifli'fv  'H  7'>  fiWn  i A.  Cii 

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'9vX^0,uoX^''  an,  ,4  <;■)  iiXeyn  ;ac£'i? 

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i»-.*w|'rfiiy 


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1. 


which  unfortunately  was  not  well  controlled.  But  an  examination 
of  other  experimental  sheets  in  the  series  shows  that  in  a num- 
ber of  cases  a definite  rise  in  the  positive  column  took  place. 
These  observations  are  summarized  in  Table  V.  It  will  be  noted 
that  in  Experiment  9 A,  the  chloroform  solution  produced  a def- 
inite rise  of  6.5  mm.  This  could  hardly  be  due  to  temperature 
althou^  this  point  needs  to  be  tested  carefully.  If  now  the 
results  are  due  to  evaporation  in  the  negative  tube,  then  tlie 
question  arises  as  to  why  evaporation  did  not  lower  the  liquid 
in  the  positive  tube  equally.  The  two  tubes  unfortunately  were 
not  always  of  equal  hei^t,  the  greatest  difference  between  them 
in  any  osmometer  being  32  mm.  In  most  cases  they  were  nearly 
equal  in  hei^t  and  in  several  cases  the  shortest  tube  was  the 
one  in  which  the  lowering  of  the  liquid  did  not  take  place.  It 
seems  very  unlikely  then  that  unequal  evaporation  was  responsi- 
ble for  the  results  obtained.  If  now  we  return  to  our  working 
hypothesis  that  the  skin  exercises  a definite  absorptive  function, 
this  phenomenon  might  be  explained  thus: 

Evaporation  takes  place  equally  in  both  tubes,  but  as 
fast  as  the  liquid  evaporates  in  the  positive  tube,  it  is  re- 
placed by  liquid  drawn  in  by  the  absorptive  force  of  the  skin, 
and  in  some  cases  this  liquid  drawn  in  exceeds  the  amount  lost 
by  evaporation  and  the  level  of  the  column  of  liquid  rises.  Thus 
the  coluxnn  in  tlie  negative  side  would  be  lowered  by  evaporation 
on  that  side  plus  the  liquid  removed  to  the  positive  tube  by  the 
absorptive  force  of  the  skin. 


'■tasrt 


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kltMh'i.f  4 ,i_  «.  ii  _ L ^ '’  •<  ■ '‘'f'  ,’ ' ' 'i 


ico  tit  .'ia£54*-.<iC^v'4>  7^;^  ci -je;^W*Lpi 


f "7^''’  ti.L-  ,t-.  fi'-x;w;4  j|  txjijvo-oj 


<noT-,  ^.;rc 


t .^J^^  "at  '4^ 


fcU- 


• A 

Hi 


t&-~  *^  ..  aii'-iit  f.-ii,  *p#  ao  •,•  «(!x  fri'»rt*t: 

a.*  j •.,vu.v  mua,,xi4iiftr^  wW«w««..' 

at/a  Oi  :r.r.a-(  W o*  4;  n0' 

,noXJ-o-.,v»  avX,tr.ooj;A  ojxaxtpc  a U VXetocv^i 

" , **  , 

^ ' •3‘*x.j  hawi^.x^.ta  •■'tf  nonfit:caaaQ'«ii(,^ 

Mv-aim  ^—1  A«*  K-.  ^ ^ 

- <.;..i  -asY.  7VX>^XaojJ:.ny  nV.eeX.<«v<l4'-tt  ►Xupt.f  «ri|A.  ■M'i''  t 

J --.^■i  9rtj  .i  i>p^Pl£  .aiM' m 

' ■ -•■■  • ' ' ■■  ' ...s 


^:»««T  „:ti  -la  eiw  b,;a  0- xd^ 

jf.  .<oit«-io"^x,y«  V a«7.?:*ox.  arf.Wirv^  i*).r  »«,  «i  Muio^ 

I e.^.  Y-  j 

i * ’ * ‘ V k^JB'  ih 


a?rearf'« 


Ir  ojt 


-20- 


More  accurate  methods  must  he  used  and  more  experimental 
data  must  he  gathered  before  we  may  consider  any  of  these  points 
as  proven,  hut  the  field  seems  to  he  a promising  one  and  worthy 
of  more  careful  investigation. 

V.  Respiration  Apparatus 

As  a convenient  index  to  the  rate  of  metaholism  of  the 
animals  experimented  upon,  I have  chosen  to  use  tlie  rate  of  CO 2 
production,  and  with  this  in  view,  a study  was  made  of  the  Biom- 
eter described  by  Tashiro  (’17)  and  of  the  titration  apparatus 
described  by  E.  J.  Lund  ( ’19).  These  were  modified  and  enlarged 
in  various  ways  to  suit  the  needs  of  the  present  study.  A des- 
cription of  these  apparatuses  as  modified  follows. 

Apparatus  for  Obtaining  COg  - Free  Air 

An  essential  part  of  any  apparatus  for  the  determina- 
tion of  CO2  is  a means  of  thorou^ily  cleaning  the  various  parts 
of  it.  This  involves  not  only  the  use  of  cleaning  fluids  but 
also  the  use  of  dry  air,  free  from  COg,  with  iwhich  the  various 
chambers  and  tubes  should  be  rinsed  between  each  two  readings. 
Figure  4,  Plate  1,  shows  a diagram  of  such  an  apparatus,  modeled 
after  that  described  by  Tashiro. 

When  a supply  of  pure  air  is  to  be  prepared  bottle  H 
is  full  of  a 20^  solution  of  NaOH,  and  bottle  F is  empty.  The 
suction  pump  attached  to  tube  A is  started,  screwcocks  D and  0 


• , r » ^ 


rx‘^42:-  riL»-r 


^ ^ J ■ ' v^. 

tnc 


ll 


1 f?M«:  o«iu/  •»j«  ruoo#.  «ioi» 

fr>.!iu(|  ©itsrU/ ^w-  us^txfQOoo  'ipa  &^^  cn^^si<f  ao 


»'.  %(t«i  r«r  «iilT'jla.c''.(?  a sHf  r5a?»«  fp“ 


7-',' 


. % 


-•  T - 

j ■ 


r f 3%;  1 si5Y<a  i!i=-: f»*pQi/ 


1 ' 


*>  . .»•>’■ 


c, 


.i>  T.0  is0  ci  j^t>rd  ." .mi{tC’V;y<^o  <?  *^V 

. ' 1 

^6  -Hi4r4^^  r.ti :■♦*;■•.  ffT»i  X ,i>  fi-'- fudm>%r»i%ii^i;|eXn4 


^«isot3  p-Y>'i>  Lur  >s,d  .-.  ,¥niv  ni''.i-'»  ^4;%..*^  * ,rc<|:; 


7. 


. foi:!  otifi  \^ 


'Si 


!>^riAlPa  b % r^^-vt  . . n * } uiukI  . \,  b ■sc^i^trtsJb  12' 


r A — ^ t t 

H ’*  ■ • .V  „ \ ,.  il  , r*.-  . i!. 


45r.  n i>  f'tfuiiuiq,.  yrViU  ^PO\tfcii^iT;5^ 


^ #» 


3niinl*5  ff<  '4(</» 


■ ./• 


->fatr*-pv  !j*v,  -r  :*o'l  sna.  .•,()  diki 


V. 


S f 


, ■■  .Wrr-Jt  Ri?‘pixDr  U'  ir/uu^'i  ^ aX.  .OCi^‘'j. 

B'  Ml  '>  ’ ,/  ■ > ® ' *1  ’ i-  '^ 

’§V  o,T.y  ori>. '\i/io- -^vX^yni  aJtrf!!®  F A^J- 


I Ot'oXtjfeV  ^ i^;Ul^^r>QD  irC'^'X  lo  ,‘5Xr?  YJI/*  Ov.ii»  lf9 

■ , J -A»  IS 

vjj'  .6.^X^Anu  o^'i  ;fs;i5  ^rt^t^*i^o{^  mi  tiU  o^f  fi<i^L^t^'  6r:  W 

Jr  / '.!J,.03rt  (W.  JJ  c**-d<t?i  .,X'f54*X^  actgg^JLU^ 

<■  ' •■?  f. ^ . *.'-  * *'  *'•  f.  ‘ S -XA 

" . '>1  i1/^i  JuT  J^lf  X»pjtf|*3U:.Jir«^  ’ .jr 

H 9XlJ-0'!f  /ft^nv  ;j 

a ■ j..  . , , * ' •'  "^  f" 

' . ,>J(tJTSi  tl  ^ ti_m  .KOait  \t}  frtlS'  'i  Xo  Xii-' 

0 btiA  a^^oco^iiiid^  6k  A 04  UM. 

■ H'^v  » • ' . . .'  ,»  ■ 


X‘. 


% 


-21- 


are  closed  and  C and  E opened,  so  that  the  suction  from  the  pump 
exhausts  the  air  in  bottle  B and  tube  R and  creating  a partial 
vacuum  in  the  bottle  F,  thus  drawing  the  NaOH  from  bottle  H thru 
the  tube  G and  into  the  bottle  F.  Bottle  H is  thus  emptied  and 
is  in  turn  filled  with  air  drawn  in  thru  tube  I (screwcock  N 
being  open  and  K closed).  This  air  has  been  sucked  thru  the 
series  of  alkaline  towers  J,  in  which  process  it  is  bubbled  thru 
the  solutions  of  NaOH  contained  in  them  and  thus  freed  from  any 
COg  it  mi^t  contain.  Thus  as  the  suction  continues,  bottle  F 
is  gradually  filled  with  NaOH  and  bottle  H is  filled  with  CO2- 
free  air  which  is  now  available  as  a supply  for  cleaning  the 
Biometer.  Ihen  bottle  H is  almost  emptied  of  its  contents  of 
NaOH  the  suction  pump  is  stopped,  screwcocks  E and  N are  closed 
ti^tly  and  the  supply  of  air  is  ready  for  use.  Part  of  the 
NaOH  from  bottle  F siphons  over  into  H but  this  soon  stops  if 
everything  is  tight.  Usually  this  air  is  forced  under  pressure 
thru  the  Biometer  and  the  method  of  procedure  is  as  follows. 

Screw- cocks  B and  E are  opened  to  allow  the  air  to 
enter  bottle  F and  then  screwcock  K (previously  closed  ti^it) 
is  opened.  The  NaOH  in  bottle  F now  siphons  thru  the  tube  G 
and  down  into  bottle  H.  The  pressure  in  bottle  H forces  the 
CO2  - free  air  contained  therein  thru  the  tube  P into  bottle  L 
where  it  is  bubbled  thru  weak  NaOH  again  as  an  added  precaution 
and  then  passes  out  thru  tube  M and  into  the  Biometer.  With 
the  bottles  F and  H of  the  proper  size  ( 10  gallons)  a continuous 
stream  of  COo  - free  air  may  be  forced  out  thru  tube  M for  more 


•.TT- 


C okutf  si.'y  ireni  ncj  j^we  »i«  >3»r«  o»  ,UB.e*qo  <i  »>#»  f jOna  fc88©l|' is® 

«■•  irjt  n Jtr»»  f tJjJvttJtl  gin  erJ  j}ow«et>v,i 

K a«>  30«ie  «!»  .7.^0*  *r..*  I' 

•i.6»  u a oijjoe  .V  8XJ.  >r  «a.'  mbx  p orfl,  ‘ * 

^ *1  4»ocarv»‘t^)  I*  8tfu?f  n|  TI*  pi  e t ; 

n<-yu  i»A*-  lia  «ixn'  j 


*■  . . .,  - ^ - ' 

. i^oI'<S^vd  ei  Ji  tti  ,.i.  ^ .. 


ijQi,  ^n  'r  r 

^ «cii5;*F  tpi  f;-;  .p-it  .riia^f^u;©.  a 

'.00  i;>,  p*:jXvCi!J  el  H i,j[  iauXi't  C.jt- 

5 I * ' . i ''  -fc  " ^ 

\ia*\te  A.  8^  cl  /i^aiw  j.lJ  tirr'i 

: 1A-.»4*!^4'«?o- ,«t1i . to  ®fl^<ir*<  ;^. »*£ . ii  7P%ir,:V„'  .•v.J.sVia 

ft  68J!CXo  PTtt,!<  pfu)  ."  d>!opo^  QtOfc  ,J  8a-3_r,j£  «i  BOiaoii*  HOija 

IL  •■  “.1  «'%»  _ 


Oi^l  ^o'lx^u  .ftiifxi  -64«*z'  «i  TfU  lo 


'li  no^ii  F^ias  iiKf  S o^ffi  ^ K?C\gM- 

.>'C.iOXC  scbxuf  ftj  >f^  -. 


'-.^'V:.iOXC  SCbxuf  8l  :tkr.  81 

.ewoIXo:  OA  8l  1®.  6^rlj<>i4  ciV 

•>  , ''  « ' ^1  ’ ' .»■/., 


w ^ ^ r , 

5-.aix  p;^4l  <xJ  la^a:»^c^  u*rj4  a J^^iJ.  U .V^l^iw-ttwtoe . 

f ■ 


, V»a^''b6«»rs  v£««eiy9-iq‘)  St-^pr(,w»5sc  xwis;  Xfj'iM  « 


® •'f-?  f.-c^i<!i..  «f&B>  oi4JM!  bX  i<Oea.UrtI  .ttBitji^gi, 

'^i‘ 

,u 


^ r.onrro^^’^  o£xi  !>’1m<? 


. a mvJ'  nlihavi  Dc^xj/tcro  {joq,\ 

»ioiiij^03’i<:  ;X^aqi>^  ^A-/f  trsds  ,^1  11  ' 

‘ -M  ■ A .*’'*»  I / 


&X1.®  C(f  oJifU  irw^xb’  hlxik  ’ 

■"  - I 


^l''  :■  s’  , ^ • V - ' i ' ' 

'tJ  AW  Ofi!  i>o«  M »<art  «-,(»  ;^i;o' uBs’exi  bob.'  SnaC 

. . ^ ‘J  X ' .'■'  ■'  ■ ' ifcS 


■cuc5>4W(T00  :,  'saoXijig,  OX  ) tislB  OKjo**  *b  I,  3 604  t' n^XJ^o’ 

. ^ . ■■  ■ , . • -80 


t 'i  virm  ‘^05  U 8rfji/? 


ifim' Aaoa^ji  0(f  93rxl:\  vOO  to  me%/i 

.r*^\  • ^ * ‘.  y ■■■-  r ■ i'«  • - ■ ' t^i  tfJTL  ' 

■>|B'  ,■  >;  ■ ,,  ^ 


»fl 


-22- 

than  an  hour.  The  time  required  to  refill  the  hot tie  H with 
pure  air  is  also  about  an  hour. 

Titration  Apparatus  for  Determining  COg 

Plate  2 represents  an  apparatus  for  the  determination 
of  CO2  by  titration,  applying  the  principle  used  and  described 
by  E.  J.  Lund  (1919),  namely,  the  titration  of  standard  HCl 
against  standard  Ba(0H)2*  The  apparatus  is  used  as  follows. 

Standard  Ba(OH)g  from  bottle  A is  admitted  thru  tube 
B into  the  side-arm  burette  C,  and  is  titrated  against  standard 
HCl  similarly  admitted  to  the  burette  D.  When  the  two  burettes 
are  filled  screw-cocks  1 and  2 are  closed.  Pinch-cock  3 is 
now  opened  and  a known  amount  of  Ba(0H)2  is  admitted  into  the 
leveling  bulb  E.  This  is  now  diluted  with  distilled  water  ad- 
mitted thru  tube  F and  the  apparatus  is  ready  for  use.  Tube  G 
is  connected  to  the  respiratory  chamber  ( to  be  described  later) 
in  which  the  animal  is  contained,  and  tube  H leads  thru  the  dry- 
ing tube  I to  the  suction  puimp.  By  means  of  this  suction  pump 
on  the  one  side  and  the  pure  air  being  admitted  under  pressure 
to  the  respiratory  chamber  on  the  other  side,  as  will  be  des- 
cribed later,  a current  of  air  is  kept  passing  thru  the  tube  G 
and  bubbling  up  thru  the  solution  in  the  bulb  E.  The  COg  con- 
tained in  the  air  unites  with  some  of  the  Ba(0H)2  to  form  an 
insoluble  precipitate  of  BagCO^  and  the  excess  Ba(OH)g  can  be 
can  be  titrated  with  standard  HCl.  After  the  experiment  has 


■ f 


n;  ir  V 4xxi«* ' 03  fe*^-?i#.7>ik*r  <Kai,3  «>t{I  iifc 

'><.  . ...  1 


^ 3‘^J-id*L.ooX'r  ti'  iJt  # y^ii/g 


V,' 


e '*^  ’■ 


»■  atJ-XiU-riN,  a/>,  i..‘j^  ,v  L ’.'.:r^AXUy:'>i  e>3  I? 

rr-3S3i%oi:of*  ^:o' |^Cp  *)i o ' 

o-j^,a.'jV^rv^>u4  * ' 

i i^:. f A i:  »tj^r  tif  MCI  1 c ( ^ • v3  - i / V n. 

*"  ^ - w - • 


9X  ^ ,-;Uico-if;orri^  «^ot  oliS  ?>ts  i'  tita^4  B dXr^^^.'li 

ofi3  tc  &iWc^  o t^wp 


otja 


-^'«  in.'sr  I’-  .tr  »rt  *il  mxy:  .V  -,r.ii^l>X 

’ ■ . ’j  ' ,^  • • •»«» 

^ n'tfp^  .e^u^'ial  ffi  er^.rj^  tv’c  VriU  'i  ^tfus  iJ:ui^M4lei 

iaftdi^oa^ll  ci;  ) ftSt3*6X  h»j^^rmi&  jttf'  * 

^ ' N .2  ‘-V 

bn«  iW'UM  | 

* ■ ' • - . • - . . - j ■ I 


^rwq  noit^sne  nit.^  \c  cii^d'a  .^tmfq  n<Uw*'Cii;a  e»iO  vi 

‘ * • 4 • ’ - ..  m'^  ' 


'*  * “ ^■***  ■ , * , J ^ ' 

-fOjjJ  9rf  ho-  \io#i>iEJ6:qtt9t;™n3l  o^ 


0 K’l  ^Ja  lo  t/tf^-ii/'o  Votf'lVo  !f  J 

* ' "J  “•  ' I* 

-itoo  ^00  eit*  hX  ox€J  e%rf3^^« 

V cr(0t  o3  c*(HC;)w^  i©  euaoQ  »»jJiiuk/  >xi4f  %fU  hi  Doi.'iai  ' *i 

-,  ■ , ■‘•‘A  -' * “ < 

U3  ;/wo,  i.( so)^. •ftfPidsa'ifi 

I , «-  ' ■ .•  i ;«  ‘ 

■«ifri  ii*urJ<tixixa  p'tat^li^\,.rsz  ini^Mte  fjtu  oJ  So^ 

i-'’'^'  ’,  ’ r-  • ' ' • i >£■  ■ * ^ 


. . .-  ^ tt  JlL<  .»IF 

. ine^  =>as'-,-7awjjw  Miinnxyji 


•23- 

"been  continued  for  an  hour  or  any  other  period,  the  suction  pump 

is  stopped,  screw-cock  No.  5 is  closed  quickly  so  as  to  catch 

% 

all  of  the  solution  in  E ( this  can  he  done  if  tube  G is  of  rather 
small  size)  and  the  solution  is  ready  to  titrate.  A few  drops 
of  phenolphthalein  indicator  are  admitted  to  the  bulb  from  the 
thistle  tube  K and  the  solution  is  titrated  with  standard  HCl 
from  burette  D.  The  HCl  used  represents  the  excess  Ba(0H)2» 
i.e.  that  which  has  not  united  with  CO2  to  form  Ba2C03.  By  sub- 
I traction  the  amount  of  Ba(0H)2  used  is  determined  and  from  this 
figure  the  amount  of  CO 2 which  has  united  to  form  Ba2C03  is  cal- 
culated. This  represents  the  amount  of  CO2  in  the  air  breathed 
out  by  the  animal  in  the  chamber  during  the  experimental  period. 

The  Respiratory  Chamber 

The  respiratory  chamber  for  use  with  the  apparatus  is 
shown  in  Big.  6,  Plate  An  ordinary  bell- jar  L with  the  tubu- 
lature  (atM)  near  its  bottom,  is  inverted  inside  a battery-jar 
N N and  covered  by  an  ordinary  desiccator  cover  0 0.  Application 
of  vaseline  around  the  edge  of  the  desiccator  cover  and  the  addi- 
tion of  a weight  of  some  size  on  top  will  usually  make  an  air- 
tight connection.  A wire  cage  P P containing  the  animal  experi- 
mented upon  is  placed  inside  the  respiratory  chamber.  The  bat- 
tery jar  N N outside  the  chamber  is  kept  filled  with  water  which 
may  be  of  any  desired  temperature  and  may  be  siphoned  in  from  a 
reservoir  above.  Thus  the  temperature  in  the  respiratory  chamber 


r 


^ • 4,.  * ■ ' ^ 


.! 


' * \ ^1 
4 


I . ■*■ : ' . ; ^ . 

ii  ..vtfft:  .nal^w»K  s*f^V' r li H&di9  xc  latiiT  iie  xo'^yUBUcd^nob  'lyi^t^ti 


i 


Mz'ihO  lij  uci  tTtriiclo  d *eIT  >l^?'’^--•4i.^Hrpi  «3t 


6- 


c -^.o  0 li  r-ac© 


r^o  9it^:t  ) L hi  tioitf Ir'jtflt  tr;  /X  /i 

9i  iittfs.rifit  i>n*  ^uitg: 

moy’'-  iSUrf  rj  Li*ttflfi8fsm  rrr«  ‘Xt' frfsI^ihqXOiWW  "t P ,#:i 


’_  (•  'i- 


h$j&^  li  Mi  r.ir*uJCo9  9'y  t>rm^ 


P . »tsj  <f  # l-rfiux.  /x^H 


F v-'-x  - sr?<'  t 'aCw  trjirtj  jf>«  rfoltii 


■ rt*li  fito*/'*  tyrtu  iibhii*r.t\*?f  nt 


r .i 


' ' .'1*-  > V l.Tii  v T^  oi 


-:v:o  .?i  ^rOOy-at'  iirica  'Of  Joo  lo  .ThzfOfi*;  ^h!f  rxua'i.'X 

- . ^-  - '■■  ' ' * .•r'4 

>'-*Si-H^*cc  ’li^T  #vfT^  ff.l  vi>.?  *tc  '£•'.•  ' J .«7^''Xi.'0 

. i . :-pjSi  ivtix'*  <>r”  M J. • vr.i rtl,  »>?»  .iftf  Jap 

;‘..r 


' ) 

-.-f 


1^1  3AC*»*x^X^/i  t^  r>:  Sc.  x xc^'Jk^'k  x%pxM.,\iiiBpx  oisT.^ 

-ucTw^  S ii-  iiJiyf  4.ir^.f,-l;^acr  ’c !/ gifoiity.  fiA  {>4^^:.i*T  ,d-  it  KWOft^ 

a . '•  ■■■  • * '"■•  „ ' <•■»  *^'-v 

^ oi.'’ ;*i  bJJ  ww?  (a  >i?) 

0 -i^r^vjpro  iii*  .y.<3'  Xic>*rgrc>o;  . |^ 

Ibpu  010  bni  »f(J  t-  0Sl-l>ci/c<'£#  «N3Uf4iiv«xo'  i!' 

-^liK  njfi  xXX-'iVf  w 1?0^  n/>  dria,  och#  ij  lo^hoi^  ' 

'•  - . <•  ■ - ■ ^ f . ' '• 

-4^^  0^T  \-Todri»nu  xwjril4iee%  e-^  e^.iehl  ' 

1 * ’ i ' - ' . ■ " --!  ' fjA 

||^:  ^ at  vh«-n<?hQlfi  ^ t>uxU\)i}  xnh  ‘to  ’c»S) 

/j  *^  . '■  ' ■ .- ' ’ ■f'l'  ' ' ’ 

50ji«Df:oy»<-Aoa^:'i:|^x.fti>  oitj-  til  -iji 

,-  . ■ ‘ . -i"-^  r r.4S' . . j"  '‘^'  ' 

' ‘ /..k  ■ *“  ' ' ■■.  . .■>'1-^*^  * 


-24- 

may  be  kept  constant  or  may  be  gradually  changed  according  to 
the  needs  of  the  experiment.  The  chamber  is  used  as  follows. 

The  wire  cage  P P containing  the  animal  is  placed  in- 
side the  respiratory  chamber  and  the  cover  0 0 is  vaselined 
and  placed  tightly  in  position,  being  held  down  by  a wei^t  if 
necessary.  CO2  - free  air  under  pressure  is  admitted  thru  the 
tube  0,,  passes  down  into  the  water  jacket  and  around  thru  the 
glass  coil  R R,  thus  being  cooled  or  warmed  to  the  same  tempera- 
ture as  the  air  in  the  chamber  and  finally  enters  the  chamber 
thru  the  inlet  at  M.  Here  it  circulates  around  the  cage  PP  and 
passes  out  thru  the  tube  T at  the  bottom,  and  thence  thru  the 
tube  G into  the  titration  apparatus.  Since  CO 2 is  heavier  than 
air,  practically  all  of  the  CO2  given  off  by  the  animal  will 
sink  to  the  bottom  of  the  chamber  and  pass  out  into  the  titra- 
tion bulb  without  difficulty.  Por  certain  experiments  it  might 
be  desirable  to  regulate  the  pressure  inside  the  respiratory 
chamber,  in  order  to  avoid  subjecting  the  animal  to  either  a 
hi^er  or  a lower  pressure  than  that  of  the  atmosphere  outside. 
This  may  be  done  by  regulating  the  screw-cock  controlling  the 
inlet  of  pure  air  on  the  one  side  and  the  suction  pump  on  the 
other  side  of  the  apparatus,  and  as  an,  indicator  of  the  pressure 
inside  the  chamber  a manometer  may  be  attached  to  the  tube  S. 

The  manometer  used  consists  simply  of  a long  glass  tube  bent 
sli^tly  in  the  middle  as  shown  in  Figure  7.  The  end  of  the 
tube  A is  attached  to  the  tube  S of  the  respiratory  chamber. 

End  B is  left  open.  Inside  the  tube  at  C is  placed  about  one  cc. 


<•>  *^5;  0<t  to  ggrcf>.' 

'■?*'  *‘* 


. *-?vO,C*  0)^  9/1  £|*1«U  ^ i TO'Pl^fU’  ^^fl?  ^rUt'^Ty^  x>rti 


I 


/. 


rr‘-^- 

•" 'j; 


^iviis,*f  ®i  gou  .^v.  n*ut<f.xii  nciTjsn»tiJ  ^.- 


fill 

pnj  'te  xxfr  ,-tt/ 


^ -O'!  iJOD/j/p  ftj  Xms^HA  uff.t  uJL<^ /|pf«  *i  '5  t'!T|J®d 

br.  1XP3-V  8i  0 0 I^^yoo  p-q  T«<^hi|if<l> 

, ^ ' ' 

Ij;.  . Ji  b£9d  ^h£^  .ec>Uii<Kj  <il  t^ec^>t\aA 

V ' . ^ ■ ■ 

«>-  »l  cTu^iioTo  Tt.-*  s*^‘^  - 203 

od^  '/*^'xfy  birt  P'vf?  ip/  ift/  T (J/t|  itwcir  09®n»pf 
- .a9<lP*6iv^^  efCt<  oi‘  Aer.*t4<W  -tc  Sf  ffftjro  nvito<f  ex«f/  ,«  ^ 

. , l^i*.^  t>.^4^4-9  «.ft^  fU  *ri* 

tf-j^WactsiO;  ii  Aa«.F  /i  i#  /tijri, 

'Xi^.;':^  - '”  ^ *?  ■ ■ ’ _ /K'**  1 " ‘ , a 

9-L,.  in.b'y  s^P**?*;  / » i^  f'^'j  ^ c T ■0du<i  if%cff9  4*6  B08iyij;{ 


■-  ) 


■ .o:U  al.rjf  . i^'itr.  txvfria?:..  er:i  n litv  ‘ .JcT  qi 

.>rq,‘>u:n  ai.^spltev^^o  /ti«4tPb  *i«l^  .K^XablXliti 


A/*f 


V'TOdaTico^X  nfj  tiOlpiJt  f>  14  iK-rXlrt^©*!  Si4^i©#-i>  00 

*3  'ra/(3‘X©  f»rf4  ‘»<ti49*!t.f»>o  64  a-^/fTQ  ,fi: 


A> 


n 


^’i3/fqaci54ji  /w/W*  , O"-^  j >'  tp 

, ■*  Om4  . S*l/JXoi7f  {130- ikOcvn-f'ev^pr  o/X  4 ^2<4  tinol>  cdT  t4c(T^^^ 

,-  04j4  «6  CU>i4fWa  ©r.4^  i>Aii  Af//«  f»/i6'0/f3  /tO  ti  o 4©xSl 

; •■  _'  ' ‘ „ ..  ' I ' , J 

'Ic  tiii  3*.  i>iV,  , otto  t:»jAc 

.8  edu4  .or?4  p4j  a©i4oison®«r  0 «h'l«rfjj 

, JcMtf  ftcfttj  8o«Xs,  >,rje/  4 I'o  >.vt<^j,  8#>itiaat;  ikofitr  wMooaSI.  <,rf#'?ii 


ntff  'tti  J>cm  '«(rr  .?  «i  r!W<-.iB  o* rti 

.■tatfci,;^  ^t:^m■^h■KPt  'gtlJ■  lo  g v»s^  «f»  0}  I«^x4ji  '4  A 

>1  ' «av  *f^  ‘ 


'•9«V-Skb/  J«ctto  tnoiicj  oi-  3 it  «)W-  »«s  mini  .»t),», n*jf  pi 

ft,  ■■  ".:s,  •■  ^.  ■■',  . v-,fi-jA.lifl| 

'■WptrsJiWE 


nw-tfli-ri  f ' tr^  • 


’(“•■t;  It 


7a  > fk< 


-25- 


of  Ellison’s  draft  gage  oil.  If  the  pressure  inside  the  respira- 
tory chamber  increases,  this  drop  of  liquid  will  be  forced  over 
toward  B and  if  the  pressure  decreases  it  will  be  drawn  toward 
A.  By  adjusting  the  air  inlet  and  the  suction  pump,  the  drop 
may  be  kept  at  the  center  C and  the  pressure  inside  the  chamber 
will  remain  constant  at  the  pressure  of  the  outside  atmosphere. 
Experience  seems  to  indicate  that  ordinarily  there  is  very  little 
danger  of  changing  the  pressure  within  the  chamber  enough  to  in- 
fluence the  metabolism  of  the  animal,  but  with  the  manometer 
attached,  the  pressure  is  easily  adjusted  and  this  possible 
source  of  error  is  eliminated. 

Experiments  with  the  Titration  Apparatus 

Table  VI  gives  the  results  of  some  preliminary  exper- 
iments performed  with  this  apparatus.  The  CO2  output  of  several 
toads  is  given  together  with  other  data  and  calculations  which 
mi^t  be  of  interest.  The  results  are  given  merely  to  illustrate 
the  use  of  the  apparatus.  The  wei^t  of  the  animal  used  should 
always  be  recorded,  together  with  the  length  of  time  during  which 
the  animal  was  kept  in  the  cage,  A record  of  the  number  of  cc. 
of  Ba(0H)2  which  were  neutralized,  i.e.  which  united  with  the 
COg  in  the  air  sample  to  produce  insoluble  BaCOj  serves  as  an 
index  to  the  relative  amount  of  CO2  produced  per  toad.  From 
this  and  the  time,  the  final  calculation,  given • in  column  7 may 
be  made,  namely:  mg.  of  CO2  per  kg.  of  body  wei^t  per  hour. 


rjt^±2?5; 


iiaBarau 


-OS- 


--ni;<I«<st  n,*  Xt.  .fi.e  ^kv  J ui-ji.  n’tiottl  j^'o 

ttiivc  iaoxo-j  Bd-  tti»  iiijjrJi'.'l*  .«oiua?Ri  X*'?;' ;*,-''^0J 

».  n»^-^b  0C(  iii«  Jt  •*g.xnitiot>  s\.  «e,n:}  o»(.*  ’lx  bnu  a btiwbj 

j - , ;auq  ;ioi;*&;/e  lufiT  ibit# , ai  'lU  j>fU  j ^j| 

. .©t^it^?;»curt3  pJ-iaicft  axf4  ’t?'  0':Mi7aoT:T  a_tr  wU'  S'iw 

i*  -04  ,2ii5WfB  Oitf  ot3jKM*tj  0;Jt  84ii- 

*ti^ca-oxw.B  .^ii  :.  t ai  rrs  M^4  * a.  trc  i tcd*4»<t^«l4  <.a*':o'AE  > : 

axJ’iacOii  xSivai}  tti  ,4^ftdoi44* 

>1 

*r< 


> ‘ 


* ■’-  -*  '•  W||  - 1 

.-•Xfjqr.s  p^ua  Tlo  «4fMB0*x  n.si*  d^vi^  IV-^XdviT  " ^ 

nJ:f  4 d4Xt>.:  s>cl,'<3u  ^ ^ 

iiOx;f»  «4ToX4^£ifj>x:«>  x>fof  *i4/,x,  xstto  d:  i-y  «X  4»o4 

■,&4r.54»«XXi  04  x-iAiftra  = ;.4t  1^4*  oif  4dglm 

ao  ^4iW  car  ..*  ;x5?wtIjwS 

:.aiiuc  <«.;«  av,0.4,m.t.'x,r»  ,U1,  TcajiBo,  >c6torcx  cc 
.?3  ao  190'euii  e.-tt  \0.1X9C>k  A Jta  na  .’tjcjl  ■>,«  XAiLa:  »d» 

^ ofiS  r.-tl.i-*' bsrbay  .o  -i  .beail-sxjfofl  .ST?-*"  ds^JUs/j^C^’oiae' lo 

:»e  ac  e^^*i  cO&^l®  flJViUosoa  0»('!,0I5  of  .V'l^-S ■ itJt  fid:  ab^(g09«j| 

flKia?  'ibcnt  beoxA<«(l'jOO  a«  j/uuKos  svUia.i  oa»  oA  .yAcai  1 

• CBIB  Cii-.«eri8 

^ .J>.  A 


, S.-00  a.o 


t?'^i 


> -26- 

In  column  8 this  figure  has  been  divided  by  the  body  weight  in 
each  case  and  the  results  so  far  as  this  table  is  concerned  seem 
to  indicate  that  the  CO2  production  bears  no  direct  relation  to 
the  body  wei^t.  In  column  3 some  notes  on  the  general  condition 
of  the  animal  at  the  time  the  analysis  was  made  are  given.  These 
twelve  analyses  were  made  during  the  fall  of  1920  using  animals 
which  had  survived  the  summer  and  were  being  kept  in  the  outdoor 
enclosure  at  the  Vivarium.  About  the  middle  of  November,  the 
last  of  the  toads  died  and  no  more  experiments  could  be  performed. 
Numbers  11  and  12  are  analyses  of  the  same  animal  on  two  succes- 
sive days  when  the  vitality  of  the  specimen  seemed  to  be  v/aning. 
The  animal  died  soon  after,  probably  on  November  15.  A marked 
decrease  in  the  CO2  output  from  this  toad  is  noticeable  on  the 
second  day.  In  these  two  experiments  the  temperature  was  con- 
trolled by  siphoning  ice-water  into  the  outer  jacket  of  the 
respiratory  chamber.  Outdoor  temperatures  on  these  two  days  were 
3.5°  C.  and  5.0°  C.  respectively.  The  temperature  in  the  water- 
jacket  was  kept  at  4.5°  C.  and  5.5°  C.  respectively.  In  experi- 
ment No.  10  the  period  used  was  20  minutes  instead  of  one  hour 
and  the  number  of  cc.  of  Ba(0H)2  neutralized  shows  a correspond- 
ingly lower  figure. 

The  Biometer 

Plate  3 represents  a modification  of  the  Biometer 
described  by  Tashiro  in  1917.  As  originally  designed  by  Tashiro 
the  apparatus  was  equipped  to  accommodate  only  very  small  animals 


."aji^sSRi  ^.— 


••ftCl* 


m 


r . ...  . • ,k  ‘ :^\rf. 

Ol  .rriV.ifjT,  >^Ocu^  (tfj'  x.i{  i:«ptf  .Bf  U CJlriJ 


- <saC'-5  '<i  oixW  *Ji*  ta*i  CB’  •-  ■■»<  ..244  tiqA  HMu  ,, 


c ; 9a  (f<t44^l^i>o^(I  ->(X''  Qtii  ts^tiylbetX  04 


,D 


t2^1:i!tu<\Q  >^(i4  f<0  o*4o/t  oacif  ^ fifi:;^Xoo  itl,  o(£4 

* ■ ■-  ' ' f 

t.  o,  :!'  .i:avt  situt^y.  ficf*  ftJt^V;£Jk'Ul  oKl?  o>l4  ' A\Ciua4fta*  W4^  t 


I 


«t;,4tifili!i,  V'$9X  'ta  «>;->  ^^4a>  i*i9V  Ott-ixX^^  ^Vif)w4  h 

— - ft 

* ' l*JlfO  »iP4:  .7l  Iv^'i:'  QOlOC  DT'*f?  w ^Uiii  i4h  #44  boVit,  u«  ikvif .ilc itfW  ' '.J 


rt44  ,'L  Bwt'vrl*  *v  .ftiOiioviV  «i4W-  4j»  ©^»»oioA€| 


j*  &9  Oil  0*’'  w'tCi^r  on  a f ib  ©^o4  u.'W  X0r4cj^i^‘ 


>» 


3’.- 


^ cy4  no  i wi  .--a;/  5 . t©  r*ii^iArc.M:o  XI 

- .irtf,  ^ c-^  >v3ti/3*«r4**  ».U  lo  x41i  *^jiv  tvojit  jsvio. 

* r * • ■ ‘ "a  ■ t 

'!  tilths ^ioi  X .^L  *f.iUu'’Vpl~  m(  \t><  oo4r,  b*i'b  axiX 

■fc*  • p >:> 

®i^4  fio  9,^Jrniii4fui  el  *t)V.^  oinU  civt;  .te  t4ij<x  sOD  efiJ  eif  e«*<H:o0b\ 

• » V ' *.  / 


“rtfo  tiSf,  ST v'4 *via4r-^;4  sfiv  qw4  ofio;14  nX'^  .'v^i^b  j^^xiftoe^W' 


4 . 4l' »Wh  X^4vQ.  ^4pf]  TR7i^;-pdl  Xu  AoJtioi-^, 


JO  • 


nrany  u>(jBb  ow4  ^0  6ia*To44ii**<'^iro4  %oot>4u0  ,7fs^iseiin  V,ioJ  ^ 

-Tni).ivy  fiX  tU/4«!;so5Ra?4  OitT  .'c-LC“ViJ ss:^liyit 
-Ix'-^ixo  ,nl  ^ J\J<e7lf4oe(fse;  *0  .ft  .0  ^e.*>  4 o 4.^031  uiti^r 


. in  Off  »no  "iOt^oJeiji  iVewfiJi'.^a  ti^rr  bii(^  -^aoi^vc.  *.44  Ci  .oft  inocT 

'■•  t‘  I ' - - ' I _ ’ . 


-DnaciftfTXorv  * ili^-Dn’o  44e^I>rx4jL’ec  .;.ai  u 


**  i ' ' I "'klJp 

♦ -*'#.  I'tX* 


.r\ 


‘I  ft 


'^^fiJOoIS  Xo  flti^^til\it>6hi  M aOAPX(.r9*X  • 

['  oxXrix^T  »i.3  if)^l««uv  vrU^lUsiXG  B/.  .mx  t-iiiCe#:Y  x<ir  ts<?Xxoasb1 
0-. IXe^  TSPV  ^i^i^dftJdsiXu^  c»4'  MW  Ao44ixi.r<je 

'•  ( .4#  *'  • ' ''^  '.. ' .,.'  . , ' . 


Tr-z.-r.  zr~. 


-: -cr  rt?w.'U 


i 


-27- 


or  pieces  of  tissue.  The  present  modification  is  the  result  of 
an  atteii^t  to  use  the  same  principles  in  the  determination  of  the 
CO2  output  from  larger  animals  such  as  toads,  etc.  The  general 
principles  of  the  "biometer  have  been  explained  by  Tashiro  in  his 
book  "A  Chemical  Sign  of  Life,”  but  in  describing  the  present 
modification  I shall  summarize  briefly  the  use  of  the  whole  appa- 
ratus. 


The  construction  of  the  respiratory  chamber  has  been 
described  elsewhere.  A few  sli^t  changes  make  it  usable  for  the 
Biometer  as  well  as  the  titration  apparatus.  To  simmarize  again 
the  use  of  the  chamber  in  its  application  to  the  present  appara- 
tus: CO2  — free  air  under  pressure  is  admitted  thru  the  tube  Q,, 

passes  thru  the  coil  R R where  it  is  cooled  or  warmed  to  the  tem- 
perature of  the  surrounding  water  jacket  and  thence  passes  thru 
the  tube  U into  the  Biometer.  This  adjustment  of  the  temperature 
of  the  pure  air  is  really  not  very  necessary  for  the  present  appa- 
ratus but  by  using  this  type  of  respiratory  chamber,  the  one 
chamber  may  be  made  to  serve  either  for  the  Biometer  or  for  the 
titration  apparatus.  Also  the  arrangement  here  shown  is  advan- 
tageous for  use  with  a smaller  respiratory  chamber  to  be  described 
later. 


The  CO2  - free  air  passes  thru  the  three-way  stopcocks 
1,  2,  and  3,  and  thru  the  tube  V into  the  respiratory  chamber  L L, 
thence  out  thru  the  tube  T.  This  is  for  the  purpose  of  thorough- 
ly rinsing  the  chamber  with  pure  air  between  each  two  readings. 
After  the  current  has  flowed  thru  the  chamber  10  or  15  minutes, 


^ 


IV  . 


'{«  i!tii<^€'t  onf  ;^^‘ .ojj^ri*i»^  ':tti  egocJuj  *10 

V:  rroJ:«.  ?if«  ni  M t(psp^&  \n^ 

._  . ’%*■« 

•?>.fi'  .'t  Xr>  , 0^  •XOg^'^al  OOTI 


• . ■ ' ■ ' . ' 

.'j/'jjca  c»*Kf  ^od^.:  *U<i  cvtc^lO'fttti 


Jfcrtcrirj-  5.l/^;iRitfi*j:4roSi  rl!  JiKa  ’ . Ic  «:i 4' 


l<JCf 


*“*91^ 

»I D,iu?  Tu  PiT«  kOi vjf  tMii^daxfe'  XXivio  X 

» "Id 


K>-r.  . 

^ ■ . '^  t. ' ‘y.  V-  '-a 

r.^r  ^ Lcr  :>AP»  t.!T 

. ^ I ” . 


■ •'■  -i  "'  *4^ 

c*-*cy  ii  >»''  C^:o  *A  .4^t»n>*|f(li^  ^'tHfJ*l^?fl^ 

n;jj.N:  ,«sr  • iw- ^ 4:«/Ji*kavi^  »•  Ia»or  a,,  “Xf^tscuf^' 


-i-t.  iyc^:  jr>frn#V:  .r- j*  iTv?  ‘heiff  j;giJ:  4.,*  :u  W •»« 

V < ^ , 


iV'-^  ^ itOJ ,^<1  *tc£  A(f  ti«  -iea*  •♦  .Ovtv  ;-a<jrJ|;. 


^CvT .' 


'i  o.-  ii'.a^aw  If'.  aoLow'b  cl  fl  XJltJD  o(£i 

♦s'-  J ■'■ 


oa(fWi*t;f  6-v*  ifeauoiTfi/a  1t<^ 

/:d  * 5ii4i  '4&*t  y\t\  Joff  vlX^i  at  *tlji  a/fe'  io 

' ' ‘ ' , . ' . ■ '^-  ’-^4 

^ ck/0  ,‘i  to  tiitjt/  ^-^luu  \i,  *!u^^^mxtji'i 

:,  • -■  , ■ • ’■  , ■ . ' . '>  • 

«4,^  ’ic'i  %u  fui>  -I alt  if»r(v^  la  cri]^  Cy'J^  v»‘4i«ft<'<(©u4£a,- 

I -’  -I  *.‘v  ’ 

~£L  V^»  P.|  e-tp^^.  *0^  .aiWJfcxtrQdi^  'rc^J^ritJrl  ^ 

p4ln:tiQal  c^  tt.il^.^8  o Uif.  aai; 

iW  A I 1 ^ 


.b"-,  . ,.  , . , v: 

, c ..aOO'ljtSu  iia-X'01/iif  titl-f  inUx  ssEr-rasij  'jX*  Sail  - kOO  od¥  ■-■' 

<-•'■  r ■•,  ■■  ■'  ■ ■'•*■>•  '■.''  ■ '■  ' »■  ■.  '2  • . 'i 

g,-  .1  t^9m'x‘-^  jrtoJaUqfw  BTj  o#.t_i  V a«tiu  op's  at6t  liiui  ,,.C  Wsi.  ,2 

10  aopx^utf,  e^y  J05!  'bx"’bXiIT  .T  orfuJ  eW  ’irrp»  Jop  POnV^J 

-**.%«*-*. *^11-  ...  * . .. 


.r>polh£^^  rfOBfi  ne‘  !V^p<f  -sld  .‘i*r^  'JjK^«wri6  a.'wv'  iirl«tili  yi^^  j 

{ ' ■''i''  r .'*-*'  ■ 

.nf^^CTJlax^  21  io  OX  •^V'^cwffo  {^rfi  J'r,iifx-*iro  oiilrf 

►t  I »,*'•.■  u . 


. f 
* j» 


-28- 

stopcock  2 is  turned  90  degrees  so  that  the  current  flows  into 
chamber  E snd  the  screw- cock  5 is  closed.  The  mercury  burette 
G is  lowered  until  the  level  of  the  mercury  in  E is  lowered  past 
the  three-way  stopcock  4.  Stopcock  4 is  now  turned  so  that  the 
air  current  flowing  into  chamber  E flows  out  thru  X,  and  the 
current  is  allowed  to  flow  for  5 or  10  minutes,  thus  thorou^ly 
rinsing  chamber  E with  pure  air.  Then  stopcock  4 is  closed  and 
the  three-way  stopcock  5 is  turned  in  such  a way  that  the  current 
flows  down  thru  tube  C and  out  at  tube  Y.  After  about  5 minutes 
the  stopcock  5 is  closed  and  the  CO2  - free  air  supply  is  shut 
off.  The  apparatus  is  now  thorou^ly  rinsed  out  and  ready  for 
use.  Screw-cock  6 is  now  closed  and  screw-cock  7 is  opened. 
Mercury  burette  K is  raised  so  as  to  fill  J with  mercury  to  the 
level  of  stopcock  3.  The  mercury  funnel  N is  now  raised,  thus  al- 
lowing the  mercury  to  fill  tube  V,  pass  beyond  the  three-way 

stopcock  3 to  the  level  Z.  (in  this  procedure  the  air  above  the 

rising  mercury  coliran  may  be  allowed  to  escape  thru  the  tube  C, 
stopcock  5 and  tube  Y.  ) Screw-cock  7 is  now  closed  tightly  and 
H is  lowered,  leaving  V full  of  mercury.  Next  the  three-way 

s topcock  4 is  turned  in  such  a way  as  to  leave  a free  passage 

from  H to  E and  the  mercury  burette  G is  raised  enough  to  fill 
the  chamber  E with  mercury  to  the  level  of  line  B.  Then  stop- 
cock 5 is  turned  so  as  to  shut  off  any  opening  out  thru  the  tube 
Y.  Next  quickly  remove  the  cover  from  the  respiratory  chamber 
and  place  the  cage  P P containing  the  animal  in  position  and  re- 
place the  cover.  The  animal  is  now  left  in  the  cage  for  thirty 


i « 


fir  oil  J’ o©  Q<?  tj^iivf^  5"  i06o^(4ii 

N , ‘I  "'fi  '■ 

^-^r-T  nl  .$  ':ioop-#c*r^  ®rxi  ? ‘tiitoBlIo 

vic'fto  '■•'^•yfivoi  lU  £ ni  srli^.  lo  it*vtiX  t>tiS  XIOthi  tot^vCi  O 


^'^.T  u6  hoiTuuv  t/oj  ?*i  »‘.  ’ ,ji  iDooqel# 

• * ■ ‘ * i 

et'iX  h.-T>‘.  ,2  jyL'o  SL  rca<fiv^ik>''^^sil  grtlwolt  TtiS -> 

yr.  - v^  - <>9K 


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-29- 


minutes  or  any  other  period  of  time  and  then  a sample  of  air  is 
removed  and  analyzed.  The  method  of  analysis  is  as  follows: 

With  stopcock  2 turned  obliquely  (i.e*  entirely  closed) 
and  stopcock  3 turned  so  as  to  connect  I with  V,  screw-cock  6 
is  opened  and  the  mercury  contained  in  tubes  V and  I is  allowed 
to  flow  downward  thru  6 and  thence  into  the  respiratory  chamber. 
The  vacuum  at  first  produced  in  tube  I is  later  replaced  by  air 
which  bubbles  past  the  falling  mercury.  Thus  tubes  V and  I are 
filled  with  air  from  the  respiratory  chamber,  i.e.  the  air  we 
wish  to  analyze.  Now  close  screw-cock  6 ti^tly  and  open  screw- 
cock  7 and  raising  the  mercury  funnel  N if  necessary  so  as  to 
fill  the  lower  part  of  tube  V with  mercury,  thus  compressing  the 
air  contained  therein.  Next  turn  three-way  stopcock  3 in  such  a 
way  that  tubes  I and  V and  burette  J are  all  in  communication. 
Mercury  burette  N is  now  slowly  raised  and  K is  simultaneously 
lowered  so  that  the  air  held  in  V under  pressure  is  forced  over 
and  into  J.  When  V is  completely  filled  with  mercury  up  to 
stopcock  3,  turn  stopcock  3 so  as  to  break  the  connection  between 
I and  J.  Now  adjust  the  mercury  burette  K so  that  the  height  of 
the  mercury  in  K shall  be  the  same  as  in  J.  This  adjusts  the 
pressure  of  the  air  in  J to  that  of  the  outside  atmosphere.  The 
sample  of  air  drawn  from  the  respiratory  chamber  is  now  contained 
in  burette  J under  atmospheric  pressure,  and  ready  to  be  tested. 

At  this  point  we  should  briefly  review  the  general 
principles  of  the  Biometer  as  described  by  Tashiro  - The  air  to 
be  analyzed  is  introduced  into  the  chamber  E.  Then  a half- 


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-30- 


saturated  solution  of  Ba(0H)2  is  allowed  to  flow  down  thru  tube 
A,  stopcock  5 and  tube  C and  Just  enou^  of  it  is  allowed  to  flow 
out  on  top  of  the  capillary  tube  at  D to  form  a hemi-spherical 
drop  at  D,  and  this  is  allowed  to  stand.  If  there  is  any  CO2 
in  the  air  in  chamber  E it  will  unite  with  the  Ba(0H)2  drop  to 
form  an  insoluble  precipitate  of  Ba2C03  which  may  be  detected 
with  the  aid  of  a hand  lens.  It  has  been  deteimined  that  the 
least  amount  of  CO2  which  will  produce  a precipitate  on  the  drop 
of  Ba(0H)2  in  ten  minutes  is  1.0  X 10“'^  grams,  and  according  to 
Tashiro  this  amount  of  CO2  will  produce  a precipitate  no  matter 
how  large  a space  the  gas  is  occupying  with  the  air.  Accordingly 
if  we  introduce  known  quantities  of  our  air  sample  into  the  cham- 
ber until  a precipitate  is  formed,  this  amount  of  air  contains 
-7 

1.0  X 10.  grams  of  CO2.  The  method  for  introducing  known  quan- 
tities of  the  sample  into  the  chamber  E is  as  follows:  The  air 
is  now  contained  in  the  upper  part  of  J and  the  mercury  in  cham- 
ber E is  at  the  level  of  line  B.  Turn  stopcock  4 so  as  to  allow 
a free  passage  from  H to  chamber  E.  Now  carefully  turn  stopcock 
5 so  as  to  allow  Ba(0H)2  to  flow  up  thru  C,  at  the  same  lower- 
ing mercury  burette  G.  7/hen  the  Ba(0H)2  solution  is  within  one 
inch  of  D,  close  stopcock  5 tightly.  Now  turn  stopcock  2 so  as 
to  allow  a free  passage  from  tube  I into  chamber  E.  (if  the 
pressures  in  chamber  E and  burette  J have  been  properly  a,djusted, 
no  change  should  take  place  in  the  heists  of  the  two  mercury 
columns,  when  this  connection  between  chambers  is  made).  Now 
read  and  record  the  hei^t  of  the  mercury  column  in  the  burette 


» ’ . I 


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-31- 


J.  Slowly  lov/er  burette  G and  simulta-neously  raise  burette  K 
until  the  mercury  column  in  J has  risen  a certain  number  of  cc. 
This  number  represents  the  number  of  cc.  of  air  introduced  into 
chamber  E,  Now  close  stopcock  2 and  very  carefully  open  stop- 
cock 5 until  a drop  of  Ba(0H)2  appears  on  the  end  of  the  tube 
at  D.  Close  5 ti^tly  and  wait  10  minutes,  for  the  appearance 
of  the  precipitate  in  the  drop.  If  at  the  end  of  this  period 
no  precipitate  of  Ba(0H)2  is  discernible  with  a hand  lens,  the 
same  process  may  be  repeated,  raising  burette  K and  lowering 
burette  G,  thus  introducing  a few  more  cc.  of  air  into  the  cham- 
ber E.  This  process  is  continued  until  sufficient  CO2  is  intro- 
duced with  the  air  to  produce  a precipitate  of  Ba2C03  on  stand- 
ing 10  minutes.  Then  the  number  of  cc.  of  air  introduced  con- 
-7 

tains  1.0  X 10  grams  of  CO2.  Knov/ing  the  capacity  of  the 
respiratory  chamber  and  the  number  of  cc.  of  air  in  it  which 
contain  this  amount  of  CO2  it  is  a simple  matter  to  calculate 
the  number  of  grams  of  CO 2 given  off  by  the  animal  in  the  ex- 
perimental period.  This  is  usually  calculated  in  terms  of  grams 
of  CO2  per  kilogram  weight  of  animal  per  hour. 

Use  of  the  Small  Respiratory  Chamber 

Figure  9 shows  a small  respiratory  chamber  for  use 
either  with  the  Biometer  or  with  the  titration  apparatus.  Such 
a chamber  mi^t  be  useful  for  studying  the  CO2  output  of  insects 


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-32- 


or  other  small  forms  which  would  not  require  the  large  chamber 
shown  in  Figure  6. 

To  use  it  in  connection  with  the  Biometer,  the  proced- 
ure is  as  follows:  Place  the  animal  in  the  small  screen  cage  C, 

which  is  fastened  to  the  glass  stopper  B’,  and  insert  the  stopper 
in  place.  With  stopcock  8 open,  lower  the  mercury  burette  F* 
until  the  height  of  the  mercury  column  in  the  chamber  A*  is  low- 
ered into  the  tube  D’.  With  stopcock  3 properly  turned  raise 
mercury  burette  K until  the  level  of  mercury  in  J is  at  Z.  Close 
stopcock  8 and  leave  the  apparatus  for  the  desired  period  of 
time.  Then  remove  a sample  of  air  for  analysis  as  follows:  Turn 
stopcock  1 so  as  to  connect  chamber  A’  with  the  tube  U’.  Turn 
stopcock  2 so  as  to  connect  tube  U’  with  I.  Thus  we  obtain  a 
continuous  passage  from  chamber  A’  to  burette  J.  Now  by  simul- 
taneously raising  burette  F’  and  lowering  burette  K a portion  of 
the  air  in  chamber  A’  is  drawn  over  into  burette  J.  Turn  stop- 
cock 2 so  as  to  sever  connection  with  chamber  A’  and  the  air 
sample  in  J is  ready  for  analysis  as  previously  described. 

To  use  chamber  A’  with  the  titration  apparatus,  turn 
stopcock  1 so  as  to  connect  chamber  A’  with  tube  U and  connect 
stopcock  8 by  rubber  tubing  with  tube  G (Plate  2,  figure  5).  CO2- 
free  air  is  now  allowed  to  pass  thru  the  temperature  control  coil 
R R (Plate  3)  up  thru  tube  U and  stopcock  1 into  chamber  A' , and 
out  thru  stopcock  8;  thence  into  the  titration  apparatus,  where 
the  method  of  analysis  is  the  same  as  that  described  previously 
in  connection  with  the  large  chamber. 


1 


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-33- 


VI . Summary 

1.  Contact  with  moist  soil  seems  to  be  absolutely  essential  to 
the  health  of  the  toad,  because: 

A.  Animals  which  fell  into  the  green-house  windov^  pits 
and  were  not  removed  were  usually  found  dead  when 
surrounded  by  dry  leaves  and  soil,  alive  if  surrounded 
by  damp  leaves  and  soil. 

B.  Animals  kept  in  the  green-house  died  if  the  leaves  and 
soil  around  than  became  dry. 

C.  Of  nineteen  toads  kept  in  cages  in  the  laboratory  v;ith- 
out  v/ater,  eleven  dried  up  and  died,  having  lost  46.7^ 
of  their  weight  on  an  average.  The  other  ei^t  dried 
up  and  became  quite  stiff  and  apparently  lifeless,  los- 
ing 36.8^  of  their  weight,  but  were  revived  later  and 
restored  to  a normal  healthy  condition  by  drenching 
them  v/ith  water. 

D.  In  all  these  cases  death  appeared  to  be  brou^t  about 
or  at  least  accompanied  by  a gradual  drying  up  and 
stiffening  of  the  animal’s  body. 

E.  Toads  kept  in  flower-pots  full  of  mud  or  in  aquaria 
with  a few  inches  of  7/ater  in  the  bottom  remained 
healthy  and  normal. 

2.  Some  sort  of  absorptive  force  of  the  skin  seems  to  be  an  im- 
portant factor  in  preserving  the  normal  water  content  of  the 
toad’s  body  because: 


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-34- 


A.  Toads  which  have  partially  dried  out  rapidly  talce  in 
water  when  placed  in  a vessel  of  it. 

B.  Toads  kept  in  mud  usually  remain  plump  and  healthy, 
"being  somewhat  swollen  "by  the  amount  of  water  imhihed. 

C.  Preliminary  experiments  on  the  absorptive  force  of  the 
toad’s  skin  seem  to  indicate  that: 

1.  In  the  live  skin  osmotic  action  goes  on  more  read- 
ily from  the  outside  of  the  skin  toward  the  inside. 

2.  VJhen  the  same  solution  is  placed  on  both  sides  of 
the  skin  a current  frequently  flows  thru  the  skin 
from  the  outside  surface  of  the  skin  toward  the 
inside  indicating  that  some  sort  of  absorptive 
force  is  operating  apart  from  the  usual  osmotic 
action  of  an  ordinary  non-living  membrane. 

3.  This  absorptive  force  is  stronger  in  skin  taken 
from  the  back  of  the  animal  than  in  that  taken 
from  the  ventral  surface. 

4.  The  absorptive  force  varies  with  the  solution  used 
and  with  the  water  content  of  the  animal  at  the 
time  when  the  skin  was  removed. 

Acknowled^nents 


My  thanks  are  due  to  Dr.  V.  E.  Shelford  for  the  original 
suggestion  of  the  problem  dealt  with  in  this  paper  and  to  Dr.  Shel- 
ford and  others  of  the  Department  of  Zoology  of  the  University  of 
Illinois  for  many  helpful  hints  and  suggestions  during  the  course 
of  the  work. 


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-3  5- 

Graphs 

In  the  following  graphs,  figures  on  the  ordinates  indi- 
cate the  number  of  millimeters  by  which  the  column  of  liquid  in 
one  tube  of  the  osmometer  exceeded  that  in  the  other  tube.  Fig- 
ures on  the  abscissas  represent  time  in  hours. 

Graph  1.  Showing  the  effect  of  ordinary  osmosis  thru  the  fresh 
toad's  skin.  Skin  taken  from  the  ventral  side  of  a toad. 

cane  sugar  solution  on  one  side  of  the  skin;  distilled 
water  on  the  other.  Curve  X shows  the  effect  when  the 
sugar  solution  was  on  the  inner  side  of  the  skin;  curve  Y 
when  the  sugar  solution  was  on  the  outer  side. 

Graph  2.  Ordinary  osmosis.  Same  as  graph  1 except  that  in  place 
of  the  sugar  solution  some  water  expelled  from  the  bladder 
of  a toad  was  used  as  the  liquid  of  greater  density. 

Graph  3.  Shov/ing  effect  when  the  same  solution  is  on  both  sides 
of  the  skin.  Different  effects  -with  different  solutions. 
Skin  removed  from  the  ventral  surface  of  the  animal. 

Graph  4.  Same  as  graph  3 except  that  the  skin  was  removed  from 
the  back  of  the  animal. 

Graphs  5,  6,«&:7.  Use  of  the  same  solution  on  both  sides  of  the 
skin.  Skins  taken  from  three  different  animals  which 

I 

varied  in  the  relative  amounts  of  water  contained  in  their  | 

[ 

bodies. 

Graphs  8 & 9.  Use  of  the  same  solution  on  both  sides  of  the  skin. 
Specimen  partially  dried  before  removing  the  skin.  (Loss 
in  wei^t  - 31  gms.).  Comparison  of  skin  taken  from  the 
ventral  surface  with  that  from  the  back  of  the  animal. 


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“40- 


Plate  1 


Plate  2 


Plate  3 


P la tes 

Apparatus  and  equipment.  Pig.  1.  Feeding  cage; 

Fig.  2.  The  flower  pot  type  of  inclosure;  Fig.  3. 
The  osmometer;  Fig.  4.  Apparatus  for  obtaining 
CO2  - free  air. 

Apparatus  for  determining  CO2  by  titration;  Fig.  5- 
The  titration  apparatus;  Fig.  6.  The  respiratory 
chamber;  Fig.  7.  The  manometer  for  regulating  the 
pressure  in  the  respiratory  chamber. 

The  biometer.  Fig.  8.  The  apparatus  as  modified 
to  accommodate  animals  as  large  as  toads;  Fig.  9. 

The  small  respiratory  chamber  for  insects,  etc. 


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-44- 


TABLE  I Determination  of  the  water  content 
before  and  after  drying, after  being 
replaced  in  water, etc. 


Max. 

Min. 

Aver 

Wt.in  gms.at  begin- 
ning of  experiment 

99*0 

52.5 

71.4 

Wt.  of  survivors 
after  52  hr s. drying 

60.2 

41  .0 

49.9 

Loss  in  gms. after 
52  hrs. 

39.5 

24.3 

30.7 

^ of  weight  lost 

58.0 

27.0 

42.0 

Wt.of  survivors 
after  drenching 

133.2 

80.9 

98.5 

dms,  of  water  taken 
in  after  drenching 

73.0 

39.9 

48.9 

% of  weight  lost 
by  8 survivors 

42.0 

27.0 

36.8 

% of  weight  lost 
by  11  dead  animals 

58.0 

37.0 

46.5 

Weight  of  1 1 dead 
animals 

62.4 

21 .8 

36. 1 

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-45- 


TABLE  II  Showing  the  method  of  recording  readings  on  the 

osmometer. Height  of  the  two  columns  of  liquid  were 
measured  in  millimeters  at  intervals. Columns  in  the 
table  marked  indicate  tubes  covered  by  skin, those 
marked  indicate  open  tubes. Differences  between 
the  two  columns  are  recorded  under  "diff." 


Date 

Time  Hrs. 

• 6 

^ NaCl 

5 ^ alcohol 

weak 

chloroform 

(1) 

(2)  (3) 

(4) 

(5) 

(6) 

(7) 

(8) 

(9) 

(10) 

(11) 

(12) 

~h 

— 

dlff . 

& 

— • 

dlff. 

► + 

diff. 

5/6 

10:30 

P.M. 

68.0 

68.0 

00 

83.5 

83.5 

00 

71.5 

71.5 

00 

7 

9:30 

A.M. 1 1 .0 

68.0 

60.0 

8.0 

81 .0 

71.0 

10.0 

72.5 

7.0 

2.5 

2:30 

P.M.  5*0 

68.0 

58.0 

10.0 

76.0 

65.0 

1 1 .0 

72.5 

68.5 

4.0 

8:30 

6 . 0 

68.0 

57.0 

1 1.0 

73.0 

58.0 

15.0 

73.0 

68,0 

5.0 

8 

9:30 

A.M. 13.0 

68.0 

56.0 

12.0 

65.0 

52.0 

13.0 

73.0 

68.0 

5.0 

1:30 

P.M.  4.0 

68,0 

55.0 

13.0 

62.0 

50.0 

12.0 

73.0 

68,0 

5.0 

8:30 

7.0 

68.5 

53.5 

15.0 

58.0 

45.0 

13.0 

73.0 

67.0 

6.0 

9 

8:00 

A.M.  11.5* 

69.0 

50.5 

19.0 

57.0 

41.5 

10.5 

73.0 

67.0 

6.0 

2:30 

P.M.  6.5 

68.5 

49.5 

19.0 

48.0 

38.5 

9.5 

73.0 

66.5 

6.5 

8:30 

6.0 

66.0 

51.0 

15.0 

44.0 

37.0 

7.0 

73.0 

66,0 

7.0 

10 

8:30 

A.M. 12.0 

61,0 

54.0 

7.0 

40.0 

34.0 

6.0 

73.0 

66.0 

7.0 

12:30 

P.M.  4.0 

60.0 

53.5 

6.5 

39.0 

32.0 

7.0 

73.0 

65.0 

8.0 

7:30 

7.0 

58.5 

53.0 

5.5 

37.0 

30.0 

7.0 

73.0 

65.0 

8.0 

11 

8:30 

A.M. 13.0 

57.0 

52.5 

5.5 

34.5 

27.0 

7.5 

72.5 

64.5 

8.0 

2:30 

P.M.  6.0 

56.0 

52.0 

4.0 

33.0 

25.0 

8.0 

72.0 

64.0 

8.0 

8:00 

5.5 

55.5 

53.0 

2.5 

32.0 

25.0 

7.0 

72.0 

65.0 

7.0 

12 

8:30 

A.M. 12.5 

55.5 

53.0 

2.5 

30.5 

23.0 

7.5 

72.0 

66.0 

6.0 

8:30 

P.M. 12.0 

54.0 

52.0 

2.0 

28.0 

20.5 

8.5 

71.5 

65.0 

6.5 

13 

8:30 

A.M. 12.0 

53.0 

51 .0 

2.0 

27.0 

18,0 

9.0 

71.0 

65.0 

6.0 

Increase  in  mm. 
over  original 

height  10  00  2.5  0 


Maximum  difference  19  15  6.5 


♦ - 


A*  r 


-46« 


TABLE 

: III 

Results  of  experiments 

on 

how  this  force 

varies  witl 

solutions  used 

,etc.  In 

ai: 

placed  on  both 

sides  of  tl 

Condition 

Source 

An 

Date 

Exp. 

Sex  of 

of 

( 

no. 

animal 

skin 

in 

1921 

-May 

2 

partially 

dried 

belly 

oui 

3 

normal 

It 

1 

4 

II 

tf 

i] 

5 

II 

li 

OU' 

6 

ij 

11 

i] 

8 

M 

black 

OU' 

9A 

partially 

belly 

OU 

dried 

9B 

partially 

back 

dried 

^ belly 

1920 

IV 

normal 

B back 

May 

C " 

partially 

A belly 

III 

dried 

B back 

C " 

A belly 

II 

completely 

B back 

dried 

C " 

(dead) 

-46- 


TABLE  III  Results  of  experiments  on  the  absorptive  force  of^  the  toad's  skin,  showing 
how  this  force  varies  with  the  condition  of  the  ahimal , source  of  the  skin, 
solutions  used, etc.  In  all  experiments  here  shown|  the  same  solution  was 


placed  on  both  sides  of  the  skin. 


1921 

- May- 


May 


Condition 

Source 

Arrangement 

Exp. 

Sex  of 

of 

of  skin 

no. 

animal 

skin 

in  osmometer 

2 

partially 

dried 

belly 

outside 

down 

3 

normal 

tf 

It 

II 

4 

» 

ft 

inside 

down 

5 

It 

if 

outside 

down 

6 

II 

n 

inside 

down 

8 

It 

black 

outside 

down 

9A 

partially 

belly 

outside 

down 

dried 

9B 

partially 

back 

If 

rt 

dried 

_A  belly 

IV 

normal 

B back 

It 

ft 

C " 

partially 

A belly 

III 

dried 

E back 

ft 

II 

C " 

A belly 

II 

completely 

B back 

If 

It 

dried 

C " 

(dead) 

;j 


Millimeters 

of  difference 

between 

the  two 

columns  of  liquid. 

Condition 

A 

B 

C 

of  skin 

■ max. effect 

max. effect 

max. effect 

when  used 

ln.6  ^ NaCl 

in  5^  alcohol 

in  weak 
chloroform 

fresh 

i 

! 0 

1.5 

1.5 

It 

in  water 

! 0 

i 

1 .0 

22.5 

10  min. 

i 1.0 

0 

1 .0 

- fresh 

ti 

1 4.0 

1 .0 

7.0 

1 1 

1 0 

1 .0 

1 .0 

It 

A fresh 

i 19.0 

I ^-5 

15.0 

6.5 

E NaCl  3hrs 

4.0 

C " 

A fresh 

1 .0  i 

35.0 

B NaCl  3hrs 

tt  It 

i ■ i 

40.0 

c 

i 

1 7.0 

2.5 

fresh 

It 

1 

' t4.5  ' 

. 24.0 

36.0 

35.0 

12.5 

37.0 

1! 

83.0 

8.0 

] 


-47- 

TABLE  IV  Showing  the  difference  in  the  osmotic  force 
exerted  by  skin  from  a normal  toad  as  con- 
trasted with  that  from  one  partially  dried; 
also  between  skin  from  the  belly  side  and 
that  from  the  back  side  of  the  animal.  Fig- 
ures represent  maximum  differences  in  mill- 
imeters between  the  two  columns  of  liquid 
in  the  osmometers  used. 

Condition  Solutions  Skin  from  Skin  from 


of  animal 

used 

belly 

back 

Experiment  no. 

5 

8 

NaCl 

4.0 

19.0 

Normal 

Alcohol 

1 .0 

15.0 

Chloroform 

7.0 

6.5 

Experiment  no. 

9A 

9B 

Partially 

NaCl 

3.5 

1 .0 

dried 

Alcohol 

4.0 

40.0 

Chloroform 

35.0 

2.5 

TABLE  V 

Showing  actual  Increases 

in  the  h( 

liquid  columns  ; 

In  various 

experimi 

Solution 

Experiment 

numbers 

3 4 5 

6 8 

9A  9B 

.6  % 

0 0 0 

0 1 . 

0 

1 .0  0 

NaCl 

0 0 0 

1.0 

0 

0 0 

5 % 

2.5  0 0 

0 

0 

t.O  0 

alcohol 

2.0  0 0 

0 

0 

0 0 

weak 

2.0  0 1.0 

0 2. 

5 

6.5  1.5 

chloroform  00  0 

0 

0 

0 0 

- t 


I 


•V  '• 


\ 


. \ . . w • : t 


t 


f 

'* 


•. 


f . ‘ 

- j 


••  I 


< 

It 


j 

t 


. '4  . 


Pi 


<« 


t/X4 


-48- 


TABLE 

VI 

GarDon  dioxide  output 

of  twelve 

toads  as 

determined 

by  the  titration  apparatus 

Date 

No. 

Condition  of  toad  Wt.of  toad 

Time  iin 

©c  N/100 

Mg. OOp 
per  kilo 

CO2  per  kg. 

in  gms. 

min. 

Ba(0H}p 

per  hr. divided 

1920 

10/30 

neutralized  per  hour 

by  body  wt. 

1 

Active 

73.2 

65 

11.89 

^ 32.69 

K' 

29.30 

.446 

2 

50.0 

60 

6.66 

.586 

3 

48.0 

60 

10.55 

48.35 

1 .007 

4 

Female, sleepy  but 

60 

9.84 

i - 

.279 

not  hibernating 

88.0 

24.60 

5 

Active 

68.0 

60 

9.63 

29.53 

.434 

11/1 

6 

Female 

1 18.0 

60 

11.38 

21  .21 

.179 

7 

Sleepy, not  in  hiber- 
nating position 

39.0 

60 

9.43 

53.19 

1.363 

5 

8 

Female 

109.0 

60 

15.37 

31.02 

.284 

0 

Sleepy, in  hibernat- 

60 

9.02 

1.180 

ing  position 

41.0 

49.40 

10 

73.0 

20 

3.08 

27.84 

.381 

13 

11 

Hibernating  position 
moved  when  touched 

40.0 

60 

8.71 

47.90 

1 . 197 

14 

12 

Same, stiffen  but  still 
alive 

40. 

60 

3.69 

20.29 

.507 

» T. 


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-49- 

Bitliography 

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1918.  Reactions  of  frogs  to  heat  and  cold.  Amer.  J. 
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1908.  The  Frog  Book.  Doubleday,  Page  & Co. 

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1911.  The  total  energy  exchanges  of  intact  cold 
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1906.  The  Biology  of  the  Frog.  Macmillan  Co.  N.  Y. 
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r 


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■3- 


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' ' ' . "3  w 

' . * . ■ t 

i'»  • . T 

M 'f^oM  , tlOb‘otiCi' 
<•*.  *-r<T^  i*<*Xi^O^.  oCl  .7/oX'-,'  • 

' * • ■'  ' 4"\\ 

. 'll* ^ , .G*  .X  * ■'  f'iV'.*** 

*-  •,%?  J >1',: 


» % 


JL'  ' 


, I 


<i  n/1  .yots:^:' 


. V :ti  ,xun 


'i 


.Y  tija  flU  ,r  i ivil-  Yo  T^.o  t<si^  aflff  • 


.'•  5 u p;  •■  lU-Wi  Y T m J..U'  Oti  ^ - .CUCit 


.Y  .V*  . oO  i (tPftr,  , v-./,ftt5jA 


'I?  , 


■u  lxi>ltt  .lo^fioO  cKt\  3CS  A 

;-*^3  ,.Ivt  .«,c;i^.  .firt9Xcw?<^!i^  £X4i<ni  %d  *^*(  j 


! V 


jlt^'  tl  Xj%t*‘Ti  ftr;  tiQUof^tx;}  tiX^  M 

^ , 4 ' .»f.vait’*,<l.-^6i*XV'««i??^  •!.  ?■ 

I*  >.i  *■■'  ' ^ 


Ji  i'  t li  ^ A>; 


-50- 


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